Method for improving solubility expression and stability of recombinant collagen

Abstract

The application discloses a method for improving the solubility expression and stability of recombinant collagen, and belongs to the technical field of genetic engineering. 10 In the method, part of proline in a repeating sequence (GPP) 10 is modified into aspartic acid and lysine. The repeating sequence of the application is suitable for various collagen domains, and the purity of the obtained collagen reaches more than 90%. Compared with collagen (V-P10BP10) composed of the repeating sequence (GPP) 10 , the yield of the collagen is increased by more than 23.6%, the thermal stability and solubility are improved, and the salt resistance is also maintained. The application improves the yield of collagen, and has a wide application prospect in the field of biological materials.

Description

Technical Field

[0001] This invention relates to a method for improving the soluble expression and stability of recombinant collagen, belonging to the field of genetic engineering technology. Background Technology

[0002] Collagen is the most abundant protein in mammals, widely distributed in skin, bones, tendons, vascular system, muscle connective tissue, etc. To date, 28 types have been discovered, mainly divided into fibrous collagen, reticular collagen, beaded filamentous collagen, anchored fibrous protein, etc. Among them, type I, type II, and type III collagen account for 80% to 90% of human collagen.

[0003] The collagen sequence consists of repeating peptide triplets (Gly-Xaa-Yaa), where Xaa and Yaa are typically proline and hydroxyproline, respectively. The structural basis of collagen is a left-handed helical conformation of polyproline type II, formed by three interlaced α-chains, i.e., a triple helix structure. This correctly folded triple helix structure is crucial for collagen to facilitate cell-cell interactions in the extracellular matrix. Studies of the collagen sequence have shown that the tight packaging of the three collagen chains, the hydrogen bonds formed between the chains, the extensive hydration network, and the high content of proline and hydroxyproline are important factors affecting the stability of the collagen triple helix structure. Due to its excellent biological properties, collagen has wide applications in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics, medical aesthetics, biomaterials, and medical devices.

[0004] Currently, based on its source, collagen is mainly divided into animal-derived collagen and recombinant collagen. Animal-derived collagen mainly comes from terrestrial and marine animals, and the extraction process is complex and poses safety risks. Recombinant collagen, on the other hand, is obtained by cloning the collagen gene into a selected expression vector, transforming it into expression cells, and finally purifying the protein. Recombinant collagen has higher purity, can be mass-produced, has less batch-to-batch variation, good water solubility, strong processability, no viral risks, and lower rejection rates. More importantly, the gene sequence in recombinant collagen can be modified to give it more biological functions, thus showing broad application prospects in tissue engineering and regenerative medicine.

[0005] Currently, Escherichia coli is a commonly used expression system for recombinant collagen. As the most thoroughly studied prokaryotic bacterium, Escherichia coli has advantages such as a clear genetic background, ease of genetic manipulation, short fermentation cycle, and high expression levels, making it the most widely used host bacterium for heterologous protein expression. However, due to the lack of post-translational modifications, recombinant collagen often fails to fold correctly into a triple helix structure.

[0006] In their previous research, the inventors' team designed collagen sequences to enable recombinant collagen to correctly fold into a triple helix structure. Qi Jingjing's master's thesis, "Design and Heterologous Expression of Biomimetic Microfibril Collagen," analyzed the α1 chain of type I collagen using protein computational analysis and thermal stability prediction, selecting predicted T... m A sequence with a temperature close to 37°C and a high tendency for triple helix was selected, and (GPP) was added to the N and C ends of the sequence. 10 The sequence forms a band-like fiber with periodic alternating light and dark stripes of varying dark stripe lengths. However, the yield of this sequence is low, and its thermal stability needs further improvement. Hu Jinyuan's paper, "Recombinant Expression and Thermal Stability Study of Proline-Rich Collagen," adds (GPP) to both ends of the collagen sequence. n (n=5,10,15), it was found that the collagen regions were intercalated at both ends by a certain length of (GPP). n Short peptides can promote collagen folding, but excessively long peptides (GPPs) are not recommended. 15 This can cause collagen regions to fail to fold correctly and form a triple helix structure; CN111333715B discloses a method for preparing type I collagen fibers, using N-terminal and C-terminal (GPP) 10 Based on the sequence, continuous Gly-Xaa-Yaa triplet collagen sequences are inserted in the middle to form band-like fibers with periodic light and dark stripes. However, existing technologies all suffer from problems such as low protein expression levels and poor stability.

[0007] Therefore, it is necessary to design a method that can increase the expression level of recombinant collagen, has high stability, and can promote the correct folding of recombinant collagen to form a triple helix structure. Summary of the Invention

[0008] To address the issues of low collagen expression levels and poor stability in existing technologies, this invention utilizes repetitive sequences (GPPs). 10 Based on this, some proline residues in the sequence are modified to be replaced with aspartic acid and lysine. The method and repetitive sequence improvement approach of this invention are universal and applicable to various collagen domains. The collagen constructed from the improved repetitive sequence has higher collagen content (GPP). 10 The constructed collagen has the advantages of increased expression, maintained or improved thermal stability, and increased solubility, while also maintaining good salt tolerance.

[0009] The first object of the present invention is to provide a collagen single chain, said single chain comprising a structure such as a first repeat sequence-collagen domain-second repeat sequence (e.g.) Figure 10(as shown); wherein, at least one of the first repeat sequence and the second repeat sequence contains an amino acid sequence as shown in SEQ ID NO.4 (abbreviated as KD2) or SEQ ID NO.5 (abbreviated as KD3); and when the first repeat sequence is SEQ ID NO.4, the second repeat sequence is SEQ ID NO.4 or SEQ ID NO.3.

[0010] In one embodiment, one of the first and second repeat sequences of the collagen single chain has an amino acid sequence as shown in SEQ ID NO.3 (abbreviated as P10).

[0011] In one embodiment, the amino acid sequence of the first repeating sequence of the collagen single chain is shown in SEQ ID NO.5, and the amino acid sequence of the second repeating sequence is shown in SEQ ID NO.3 (abbreviated as P10);

[0012] Alternatively, the amino acid sequence of the first repeat sequence of the collagen single chain is shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is shown in SEQ ID NO.4;

[0013] Alternatively, the amino acid sequence of the first repeat sequence of the collagen single chain is shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is shown in SEQ ID NO.5;

[0014] Alternatively, the first and second repeat sequences of the collagen single chain are both as shown in SEQ ID NO.4.

[0015] The core of this invention lies in the design of repetitive sequences, which can be applied to different collagen domains and can produce one or more of the following effects: increased expression level, increased stability, and increased solubility. Optionally, the collagen domain can be any collagen domain sequence derived from human collagen or bacterial collagen; for example, the collagen domain sequence can be the collagen domain sequence disclosed in CN111333715A or the collagen domain sequence disclosed in CN 117186210 A.

[0016] In one embodiment, the amino acid sequence of the collagen domain is as shown in SEQ ID NO. 16-22; or it is obtained by combining any two amino acid sequences from SEQ ID NO. 16-18; or it is obtained by repeating any amino acid sequence shown in SEQ ID NO. 16-22 2-3 times. Optionally, the amino acid sequence of the collagen domain is obtained by splicing SEQ ID NO. 16 and SEQ ID NO. 17 in sequential order, or by splicing two SEQ ID NO. 17 sequences.

[0017] In one embodiment, the amino acid sequence of the collagen domain is shown in SEQ ID NO.2.

[0018] In one embodiment, the N-end of the first repeating sequence further contains a folded region, with the structure as follows: Figure 9 As shown.

[0019] In one embodiment, the folded domain is connected to the first repeat sequence via an enzyme cleavage site; optionally, the enzyme cleavage site is LVPRGSP.

[0020] In one embodiment, the amino acid sequence of the folded domain is shown in SEQ ID NO.1.

[0021] In one embodiment, a glycine residue may be added to the end of the amino acid sequence of the collagen single chain.

[0022] In one embodiment, the amino acid sequence of the collagen single chain is as shown in any one of SEQ ID NO. 6 to 9.

[0023] A second object of the present invention is to provide a gene encoding any of the above-mentioned collagen single chains.

[0024] The present invention also provides plasmids or cells carrying the above-mentioned genes.

[0025] In one embodiment, plasmids of the pColdIII series or pET series are included.

[0026] In one implementation, the plasmid is pColdIII.

[0027] In one embodiment, the cells are Escherichia coli, including E. coli BL21, E. coli BL21(DE3), E. coli JM109, E. coli DH5α, or E. coli TOP10.

[0028] In one implementation, the host cell is E. coli BL21(DE3).

[0029] A third objective of this invention is to provide a method for improving collagen stability by linking a first repeat sequence to the N-terminus of the collagen domain and a second repeat sequence to the C-terminus, constructing a structure of first repeat sequence-collagen domain-second repeat sequence (e.g., ...). Figure 10 The collagen single chain (as shown); wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0030] This invention also provides a method for improving the solubility of collagen, wherein a first repeat sequence is attached to the N-terminus of the collagen domain and a second repeat sequence is attached to the C-terminus, constructing a structure of first repeat sequence-collagen domain-second repeat sequence (e.g., ...). Figure 10 The collagen single chain shown; wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0031] This invention also provides a method for increasing collagen production, wherein a first repeat sequence is attached to the N-terminus of the collagen domain and a second repeat sequence is attached to the C-terminus, constructing a structure of first repeat sequence-collagen domain-second repeat sequence (e.g., ...). Figure 10 The single chain of collagen (as shown) is expressed by a host cell; wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0032] The fourth objective of this invention is to provide a collagen protein composed of three structures: a first repeat sequence - a collagen domain - a second repeat sequence (e.g., ...). Figure 10 The collagen single chain (as shown) is composed of collagen; wherein, at least one of the first repeat sequence and the second repeat sequence contains an amino acid sequence as shown in SEQ ID NO.4 (KD2) or SEQ ID NO.5 (KD3).

[0033] In one embodiment, the collagen single chains are coiled around a common central axis, forming a triple helix structure.

[0034] In one embodiment, the collagen is obtained by expressing the above-mentioned collagen single chain in a host and then removing the folding domain by enzymatic cleavage.

[0035] In one embodiment, the amino acid sequences of the first and second repeat sequences of the collagen single chain are both as shown in SEQ ID NO.4; or, the amino acid sequence of the first repeat sequence of the collagen single chain is as shown in SEQ ID NO.5, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.3; or, the amino acid sequence of the first repeat sequence of the collagen single chain is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.4; or, the amino acid sequence of the first repeat sequence of the collagen single chain is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.5.

[0036] The present invention also provides collagen fibers formed by the above-mentioned collagen polymer self-assembly.

[0037] In one embodiment, the folded domain and the first repeat sequence are linked by an enzyme cleavage site. Alternatively, the folded domain and the first repeat sequence are linked by LVPRGSP.

[0038] The present invention also provides the application of the above-mentioned collagen single chains or collagen or collagen fibers in the preparation of collagen products.

[0039] The present invention also provides a collagen product, wherein the product contains any of the above-mentioned collagen single chains or collagen or collagen fibers.

[0040] In one embodiment, the collagen product also contains vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, natural plant extracts, etc.

[0041] The present invention also provides the application of the above-mentioned collagen single chains or collagen or collagen fibers in the preparation of collagen products.

[0042] In one embodiment, the collagen product is a cosmetic, food, or pharmaceutical.

[0043] In one embodiment, the collagen product may be a drug carrier, a medical product, or a cosmetic product.

[0044] Beneficial effects:

[0045] This invention improves the repetitive sequences in collagen expression by modifying some proline in the sequence into aspartic acid and lysine.

[0046] The method of this invention is universal and applicable to various collagen domains. This invention utilizes the modified repetitive sequence and different collagen domains to construct multiple collagen single chains and achieve their expression. The results showed that the expression levels were all improved. The purified collagen obtained after enzymatic digestion exhibited characteristics such as maintained or improved stability, increased solubility, and good salt tolerance.

[0047] This invention utilizes the method of modifying repetitive sequences to obtain multiple collagen proteins. The purity of the constructed collagen proteins reaches over 90%, which is higher than that of unmodified collagen proteins derived from (GPP). 10 Compared to the collagen composition (V-P10BP10), the yield is increased by more than 23.6%, the heat resistance temperature is increased by 2℃, the solubility is improved, and at the same time, it maintains good salt resistance. Attached Figure Description

[0048] Figure 1This is an SDS-PAGE image of the supernatant collected after cell lysis and centrifugation; in the image, 1 to 5 correspond to the sequences V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3, respectively. The arrows represent the target bands; M: protein marker.

[0049] Figure 2 The image shows an SDS-PAGE of the target protein eluted at a concentration of 400 mM imidazole. In the image, 1 to 5 correspond to the sequences V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3, respectively. The arrows represent the target bands; M: protein marker.

[0050] Figure 3 This is a graph showing the production of collagen with different sequences.

[0051] Figure 4 This is an SDS-PAGE image of collagen with V-domain after trypsin digestion. In the image, 1 to 5 correspond to the sequences P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3, respectively. The arrows represent the target bands; M: protein marker.

[0052] Figure 5 To verify whether the values ​​are the target protein molecular weights using MALDI-TOF, AD values ​​are KD3BP10, P10BKD2, KD2BKD2, and P10BKD3.

[0053] Figure 6 The full wavelength spectrum, thermochromic curve, and first derivative plot of the thermochromic curve of the designed type I collagen are shown.

[0054] Figure 7 The full wavelength spectrum, thermochromic curve, and first derivative plot of the thermochromic curve of the designed type I collagen at 0mM, 10mM, and 100mM are shown. AD represents KD3BP10, P10BKD2, KD2BKD2, and P10BKD3.

[0055] Figure 8 To prepare a sample with a concentration of 2 mmol / L, it was dissolved in 10 mM sodium phosphate buffer, incubated at 4°C for 1 day, and then photographed and recorded.

[0056] Figure 9 This is a schematic diagram of the collagen structure.

[0057] Figure 10 This is a schematic diagram of the collagen structure after enzymatic cleavage to remove the folded domains. Detailed Implementation

[0058] Culture medium:

[0059] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0;

[0060] TB medium (g / L): tryptone 12, yeast extract 24, glycerol 4 mL, KH2PO4 2.3 L, K2HPO4 12.5 L. Culture method (shake flask fermentation):

[0061] Aspirate 50 μL of bacterial culture from the glycerol tube containing the target gene into 5 mL of LB (Amp resistant) and incubate overnight at 37°C and 200 rpm. Transfer 1% of the culture to 100 mL of TB fermentation broth (Amp resistant) and incubate at 37°C and 200 rpm for 24 h. Add IPTG to a final concentration of 1 mmol / L and ferment at 25°C and 200 rpm for 10 h, then transfer to 15°C for another 14 h.

[0062] Protein purification methods:

[0063] After fermentation, the bacterial culture was collected, centrifuged at 10,000 rpm for 5 min at 4 °C, the supernatant was discarded, and the bacterial precipitate was collected. After disruption, the precipitate was centrifuged at 10,000 rpm for 20 min at 4 °C and filtered through a 0.45 μm aqueous filter membrane. Then, it was filtered using His Trap. TM HP affinity purification was performed using 5 mL of buffer A (20 mmol / L Na₂HPO₄, 20 mmol / L NaH₂PO₄, 500 mmol / L NaCl, 10 mmol / L Liminazole, pH 7.4), followed by loading at a flow rate of 2.5 mL / min. After loading, a gradient elution was performed using elution buffer B (20 mmol / L Na₂HPO₄, 20 mmol / L NaH₂PO₄, 500 mmol / L NaCl, 500 mmol / L Liminazole, pH 7.4) to obtain the target protein. The purification status was analyzed using SDS-PAGE.

[0064] Trypsin digestion:

[0065] The purified collagen was dissolved in water to a concentration of 2 mg / mL, and trypsin at a concentration of 2.5 g / L was added at a molar ratio of 10:1. The mixture was digested in a shaker at 25°C for 20 h, and the purity was verified by SDS-PAGE analysis.

[0066] Desalination freeze-drying method:

[0067] The enzyme digestion reaction product was desalted using HiTrap Desalting with ultrapure water as the mobile phase at a flow rate of 5 mL / min. The peak sample was collected, verified by SDS-PAGE, and then freeze-dried under vacuum at -50 °C.

[0068] Identification of the triple helix structure and stability of the samples:

[0069] Circular dichroism (CD) chromatography was used for identification. The specific steps were as follows: the lyophilized sample was dissolved in 10 mmol / L, pH 7.4 sodium phosphate buffer to a concentration of 1 mg / mL, equilibrated at 4°C for 24 h, and then subjected to CD chromatography. The full wavelength range (190-260 nm) was measured at 4°C with 1 nm intervals, and the average scan time was 5 s. The thermal curve was obtained by monitoring the CD signal at 225 nm, increasing the temperature from 10°C to 80°C at a rate of 10°C / h, equilibrating for 8 s at each temperature, and determining the melting temperature (T). m The stability of the sample is obtained by taking the median absorbance value of the fitted thermal curve at 10℃ and 80℃.

[0070] Protein purity determination:

[0071] ImageJ was used to analyze the SDS-PAGE electrophoresis image, and the ratio of the gray value of the target band to the gray value of the lane containing the target band was calculated to obtain the protein purity.

[0072] Methods for determining molecular weight using MALDI-TOF-MS (ultrafleXtreme) mass spectrometry:

[0073] The lyophilized samples were dissolved in water to prepare 1 mg / mL solutions, and their molecular weights were determined using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, combined with ethanol and diammonium hydrogen citrate), and the operation was performed in linear mode.

[0074] Example 1: Design of collagen sequences

[0075] Design as Figure 9 The collagen amino acid sequence is shown. The amino acid sequence of at least one of the first or second repeat sequences is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0076] The introduction of the folding domain assists collagen in folding to form a triple helix structure. Optionally, the folding domain is a V-domain.

[0077] The introduction of the collagen domain (B for short) involves analyzing the α1 chain of type I collagen using protein computational analysis and thermal stability prediction, selecting predicted T... m Sequence B, with a value close to 37°C and a high tendency for triple helices.

[0078] The introduction of the repeating sequence module can assist in the folding of the collagen triple helix and improve its thermal stability. The repeating sequences are selected from P10, KD2, and KD3; where P10 is (GPP). 10 (Amino acid sequence is SEQ ID NO.3); KD2 is (GPP) 10 The four proline residues in KD2 are mutated to two aspartic acid residues and two lysine residues. The amino acid sequence of KD2 is shown in SEQ ID NO.4; KD3 is a GPP-containing compound. 10 The six proline residues in the collagen were mutated to three aspartic acid residues and three lysine residues. The amino acid sequence of KD3 is shown in SEQ ID NO.5. Considering that the proline residue at the end of the sequence may be detrimental to protein expression, an additional glycine residue was added to the end of the collagen amino acid sequence.

[0079] Based on the position of the repeat sequence, it is divided into first repeat sequence and second repeat sequence. An enzyme cleavage site (such as LVPRGSP) is added between the fold domain and the first repeat sequence to facilitate subsequent enzyme cleavage to remove the fold domain.

[0080] Specifically, in such Figure 9 The naming rules for the collagen single chains shown are as follows:

[0081] Taking V-KD3BP10 as an example, "V" refers to the folding domain (amino acid sequence as shown in SEQ ID NO.1); "B" is the collagen domain (amino acid sequence as shown in SEQ ID NO.2); "KD3" before the collagen domain "B" indicates the amino acid sequence of the first repeat sequence, and "P10" after the collagen domain "B" indicates the second repeat sequence, where P10 is (GPP). 10 The amino acid sequence is shown in SEQ ID NO.3.

[0082] By replacing the first and / or second repeat sequences corresponding to V-KD3BP10, the corresponding collagen single-chain sequences V-P10BKD2, V-KD2BKD2, and V-P10BKD3 are constructed. The amino acid sequences of V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BKD3, and V-P10BP10 are shown in SEQ ID NO. 6–10, respectively.

[0083] Example 2: Construction of recombinant plasmids and recombinant bacteria

[0084] (1) Construction of recombinant plasmids

[0085] Starting from the nucleotide sequences of the repeating amino acid sequences V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BP10, and V-P10BKD3 obtained in Example 1 (nucleotide sequences shown in SEQ ID NO. 11-15), when synthesizing the nucleotide sequence of the protein single strand, the base GC was introduced at the 5' flanking end, and Nco I and Bam HI restriction sites were introduced at the 5' and 3' ends, respectively. The nucleotide sequence of the Nco I restriction site is CCATGG, and the nucleotide sequence of the Bam HI restriction site is GGATCC. The synthesized genes were inserted between the Nco I and Bam HI sites of the pColdIII plasmid (purchased from Genewiz) to obtain recombinant collagen protein plasmids expressing different repeating amino acid sequences. Sequencing yielded the correct recombinant plasmids.

[0086] (2) Construction of recombinant strains

[0087] The recombinant plasmids that were correctly sequenced in step (1) were transformed into E. coli BL21(DE3) competent cells, plated on LB plates containing ampicillin, cultured and screened, and preserved in glycerol tubes to obtain recombinant bacteria containing recombinant collagen, which were named E. coli BL21(DE3)-V-KD3BP10, E. coli BL21(DE3)-V-P10BKD2, E. coli BL21(DE3)-V-KD2BKD2, E. coli BL21(DE3)-V-P10BKD3, and E. coli BL21(DE3)-V-P10BP10, respectively.

[0088] Example 3: Expression, purification, and enzyme digestion of collagen sequences

[0089] The recombinant bacteria E. coli BL21(DE3)-V-KD3BP10, E. coli BL21(DE3)-V-P10BKD2, E. coli BL21(DE3)-V-KD2BKD2, E. coli BL21(DE3)-V-P10BKD3, and E. coli BL21(DE3)-V-P10BP10 obtained in Example 2 were subjected to shake-flask fermentation. After cell collection, lysis, and centrifugation, the SDS-PAGE results of the supernatant were as follows: Figure 1 As shown. The supernatant is retrieved using His Trap. TM HP 5 mL was used for affinity purification, and samples were collected at imidazole concentrations of 175 mmol / L and 400 mmol / L. The samples were verified as the target protein by SDS-PAGE, and the results are as follows. Figure 2 As shown.

[0090] The validated protein was desalted, freeze-dried, and then weighed to obtain its yield. The results are as follows: Figure 3 As shown, the yields of recombinant collagen V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 were 55.02 mg / L, 83.25 mg / L, 68.02 mg / L, 73.51 mg / L, and 91.98 mg / L, respectively. The yields of the modified collagen V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 were increased by 23.6% to 67.18% compared to V-P10BP10, with V-P10BKD3 showing the highest yield.

[0091] To remove the folded domain, a trypsin restriction site, LVPRGSP, was introduced between the repeat sequence and the folded domain during sequence design. The purified collagen was dissolved in water to a concentration of 2 mg / mL and digested with trypsin (purchased from Shanghai Titan Technology Co., Ltd.) at a molar ratio of 10:1 to obtain the pure collagen domain structure.

[0092] Under the action of trypsin, the V-domain is digested into multiple short peptides containing 2-20 amino acid residues. If the collagen domain folds correctly into a rigid triple helix structure under the action of the V-domain, it will not be digested by trypsin in a short time. After enzymatic digestion, desalting is performed to remove the short peptides, finally yielding collagen P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3. SDS-PAGE analysis confirmed that... Figure 4 As shown, the purity is greater than 90%.

[0093] Example 4: MALDI-TOF Identification

[0094] Collagen KD3BP10, P10BKD2, KD2BKD2, and P10BKD3, after enzymatic digestion and desalting in Example 3, were lyophilized. The lyophilized samples were then dissolved in water to a concentration of 1 mg / mL, and their molecular weights were determined using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, combined with ethanol and diammonium hydrogen citrate), and the operation was performed in linear mode. The results confirmed that the obtained collagen molecular weights matched the theoretical values. Figure 5 As shown.

[0095] Example 5: Circular dichroism characterization of collagen and its sequence structure

[0096] To confirm the secondary structure of the collagen domain, the lyophilized collagen samples (P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3) from Example 3, after enzymatic digestion and desalting, were prepared into a 1 mg / mL solution using 10 mmol / L sodium phosphate buffer and equilibrated at 4°C for 24 h. After equilibration, full-wavelength scanning was performed using circular dichroism spectroscopy.

[0097] The results are as follows Figure 6 As shown, P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 all exhibit characteristic positive absorption peaks at 225 nm, indicating that all six collagen proteins correctly folded into a triple helix structure with the assistance of the V-domain. The thermochromatograms at 225 nm from 4℃ to 80℃ were monitored using circular dichroism spectroscopy, and the Tg of collagen was obtained by taking the first derivative of the thermochromatograms. m Value, T of P10BP10 m The value is 35℃, and the remaining sequences T m The values ​​were all 37℃.

[0098] The above results demonstrate that the collagen sequences designed in this invention can all correctly fold into triple helical structures, compared to the T-structure of the P10BP10 protein sequence. m Increasing the temperature by 2°C improves the thermal stability of the designed sequences.

[0099] Example 6: Salt tolerance test of collagen with different repeating sequences

[0100] To verify the stability of different collagen sequences in high-concentration salt solutions, collagen sequences KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 prepared in Example 3 were lyophilized and dissolved in 10 mmol / L sodium phosphate buffer to prepare a 1 mg / mL solution. Then, 100 mmol and 200 mmol of sodium chloride were added to the solution, and the results are as follows. Figure 7 As shown, the thermal change curves of collagen from 4℃ to 80℃ at 225nm were monitored using circular dichroism spectroscopy. The T value of collagen was obtained by taking the first derivative of the thermal change curves. m The values ​​and results show that the T values ​​of each sequence under high salinity conditions are... m The temperature value remained at 37℃, indicating that the modified collagen still exhibits good salt tolerance.

[0101] Example 7: Repeated sequences enhance collagen domain protein solubility

[0102] To verify the solubility of different collagen sequences, lyophilized samples of collagen P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 prepared in Example 3 were dissolved in 10 mmol / L sodium phosphate buffer to prepare a solution with a sample concentration of 2 mmol / L. The solution was then incubated at 4°C for one day. The results are as follows: Figure 8 As shown, P10BP10 precipitated, while the other proteins did not. This indicates that the modified collagen, at the same concentration, exhibits lower turbidity and increased molecular solubility compared to sequence P10BP10.

[0103] Example 8: Application of repetitive sequences in improving collagen production, heat resistance, and salt resistance in different collagen domains

[0104] Based on Example 1, the collagen domain shown in SEQ ID NO. 2 was replaced with the collagen domain (denoted as B') of SEQ ID NO. 16-22, respectively, to obtain multiple collagen single chains. These chains were then expressed and purified to analyze the versatility of the repetitive sequence modification method of the present invention in improving collagen yield, heat resistance, and salt tolerance. Simultaneously, recombinant collagen was prepared and purified according to the methods in Examples 2-3, and the yield, heat resistance, and salt tolerance of different recombinant collagens were tested according to the methods in Examples 4-7. The results showed that replacing the collagen domains with KD3B'P10, P10B'KD2, KD2B'KD2, and P10B'KD3 resulted in a higher yield (GPP) during recombinant expression. 10 Increased by more than 15%; the heat resistance temperature T of purified collagen m It can be increased by about 2°C or kept at about 37-38°C while maintaining good salt resistance.

[0105] In addition, a new collagen domain (HC1-12) obtained by splicing SEQ ID NO.16 and SEQ ID NO.17 in sequence, and a new collagen domain (HC1-22) obtained by splicing two SEQ ID NO.17s, were also used to construct collagen single chains, express collagen, and purify collagen using the same method. The results showed that the yield of recombinant expression was also higher than that of GPP. 10 Increased by more than 18%; the heat resistance temperature T of purified collagen m It can be increased by about 2°C or kept at about 37-38°C while maintaining good salt resistance.

[0106] The sequence involved in this invention:

[0107] SEQ ID NO.1: Amino acid sequence of V-domain

[0108] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD

[0109] SEQ ID NO.2: Amino acid sequence of collagen domain B

[0110] GFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGA AGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPS

[0111] SEQ ID NO.3: (GPP) 10 amino acid sequence

[0112] GPPGPPGPPGPPGPPGPPGPPGPPGPPGPP

[0113] SEQ ID NO.4: Amino acid sequence of KD2

[0114] GPPGPPGPKGDPGPPGPPGPKGDPGPPGPP

[0115] SEQ ID NO.5: Amino acid sequence of KD3

[0116] GPPGPKGDPGPPGPKGDPGPPGPKGDPGPP

[0117] SEQ ID NO.6: Amino acid sequence of V-KD3BP10

[0118] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPKGDPGPPGPKGDPGPPGPKGDPGPPGFPGERG VQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG

[0119] SEQ ID NO.7: Amino acid sequence of V-P10BKD2

[0120] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPG

[0121] Amino acid sequence of V-KD2BKD2 for SEQ ID NO. 8

[0122] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPG

[0123] Amino acid sequence of V-P10BKD3 for SEQ ID NO. 9

[0124] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPKGDPGPPGPKGDPGPPGPKGDPGPPG

[0125] Amino acid sequence of V-P10BP10 for SEQ ID NO. 10

[0126] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG

[0127] SEQ ID NO. 11: Nucleotide sequence of V-KD3BP10

[0128] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTAAAGGTGATCCTGGTCCTCCGGGTCCTAAAGGTGATCCTGGTCCTCCTGGTCCTAAAGGTGATCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCACCTGGTCCTCCGGGTCCTCCGGGCCCTCCTGGCCCGCCTGGTCCGCCTGGTCCACCGGGTCCTCCTGGT

[0129] Nucleotide sequence of SEQ ID NO. 12: V-P10BKD2

[0130] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCAAAAGGTGATCCGGGTCCTCCGGGCCCTCCTGGCCCGAAAGGTGATCCTGGTCCACCGGGTCCTCCTGGT

[0131] Nucleotide sequence of SEQ ID NO. 13: V-KD2BKD2

[0132] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTAAAGGTGATCCGGGTCCTCCAGGTCCGCCTGGTCCTAAAGGTGATCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCAAAAGGTGATCCGGGTCCTCCGGGCCCTCCTGGCCCGAAAGGTGATCCTGGTCCACCGGGTCCTCCTGGT

[0133] Nucleotide sequence of SEQ ID NO. 14: V-P10BP10

[0134] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCACCTGGTCCTCCGGGTCCTCCGGGCCCTCCTGGCCCGCCTGGTCCTCCTGGTCCACCGGGTCCTCCTGGT

[0135] Nucleotide sequence of SEQ ID NO. 15: V-P10BKD3

[0136] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTAAAGGTGATCCTGGTCCTCCGGGTCCTAAAGGCGATCCTGGCCCGCCTGGTCCTAAAGGTGATCCGGGTCCTCCTGGT

[0137] Amino acid sequence of HC1-1 of SEQ ID NO. 16

[0138] GARGLPGTAGLPGMKGHRGFPGERGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGP RGPQGPPGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKR

[0139] SEQ ID NO.17: Amino acid sequence of HC1-2

[0140] GFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGA AGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPS

[0141] SEQ ID NO.18: Amino acid sequence of HC1-3

[0142] GPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGFPGERGAPGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQ

[0143] SEQ ID NO.19: Amino acid sequence of HC2A

[0144] GLTGPAGEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGDRGEAGA QGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPS

[0145] SEQ ID NO.20: Amino acid sequence of HC3A

[0146] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGSPGP KGDKGEPGPPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEP

[0147] SEQ ID NO.21: Amino acid sequence of HC3B

[0148] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGAKGE PGPRGERGEAGIPGVPGAKGEDGKPGEPGPKGDAGAPGAPGPKGDAGAPGER

[0149] SEQ ID NO.22: Amino acid sequence of HC3C

[0150] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGAKGE PGPRGERGEAGIPGVPGAKGEDGRDGNPGSDGLPGRDGSPGPKGDRGENGSP

[0151] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A collagen single chain, characterized in that, The single-chain structure is a first repeat sequence-collagen domain-second repeat sequence; wherein, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5, and the amino acid sequence of the collagen domain is as shown in SEQ ID NO.2 or SEQ ID NO.16~22; The amino acid sequence of the first repeating sequence of the collagen single chain is shown in SEQ ID NO.5, and the amino acid sequence of the second repeating sequence is shown in SEQ ID NO.3; Alternatively, the amino acid sequence of the first repeat sequence of the collagen single chain is shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is shown in SEQ ID NO.5; Alternatively, the first and second repeat sequences of the collagen single chain are both as shown in SEQ ID NO.

4.

2. The collagen single chain according to claim 1, characterized in that, The N-terminus of the first repeating sequence also contains a folded domain, the amino acid sequence of which is shown in SEQ ID NO.

1.

3. The collagen single chain according to claim 1, characterized in that, The folded domain is connected to the first repeat sequence via an enzyme cleavage site, which is LVPRGSP.

4. A gene encoding a single strand of collagen as described in any one of claims 1 to 3.

5. A plasmid carrying a gene encoding a single strand of collagen as described in any one of claims 1 to 3.

6. A cell carrying a gene encoding a single strand of collagen as described in any one of claims 1 to 3.

7. A method for improving the stability, solubility, or yield of collagen, characterized in that, A first repeat sequence is attached to the N-terminus of the collagen domain, and a second repeat sequence is attached to the C-terminus to construct a collagen single chain with the structure of first repeat sequence-collagen domain-second repeat sequence, which is then expressed through host cells. Wherein, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5, and the amino acid sequence of the collagen domain is as shown in SEQ ID NO.2 or SEQ ID NO.16~22; The amino acid sequence of the first repeating sequence of the collagen single chain is shown in SEQ ID NO.5, and the amino acid sequence of the second repeating sequence is shown in SEQ ID NO.3; Alternatively, the amino acid sequence of the first repeat sequence of the collagen single chain is shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is shown in SEQ ID NO.5; Alternatively, the first and second repeat sequences of the collagen single chain are both as shown in SEQ ID NO.

4.

8. A type of collagen, characterized in that, It consists of a single protein chain with a structure of first repeat sequence-collagen domain-second repeat sequence; Wherein, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5, and the amino acid sequence of the collagen domain is as shown in SEQ ID NO.2 or SEQ ID NO.16~22; The amino acid sequence of the first repeating sequence of the collagen single chain is shown in SEQ ID NO.5, and the amino acid sequence of the second repeating sequence is shown in SEQ ID NO.3; Alternatively, the amino acid sequence of the first repeat sequence of the collagen single chain is shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is shown in SEQ ID NO.5; Alternatively, the first and second repeat sequences of the collagen single chain are both as shown in SEQ ID NO.

4.

9. Collagen fibers formed by the self-assembly of collagen polymers as described in claim 8.

10. The use of any one of the collagen single chains of claims 1 to 3, or the collagen protein of claim 8, or the collagen fiber of claim 9, in the preparation of collagen products.

11. A collagen product, characterized in that, The product contains any one of the collagen single chains of claims 1 to 3, or the collagen of claim 8, or the collagen fiber of claim 9.

12. The collagen product according to claim 11, characterized in that, The collagen product is a cosmetic, food, or drug.

13. The collagen product according to claim 11, characterized in that, The collagen product is a drug carrier, medical product, or beauty product.

14. The collagen product according to claim 11, characterized in that, The collagen product also contains one or more of the following: vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, and natural plant extracts.

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