CN115850525A - A kind of recombinant human collagen and its preparation method and application - Google Patents

Abstract

The invention provides a recombinant human collagen and a preparation method and application thereof, wherein the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide; the invention also provides a preparation method and application of the recombinant human collagen. The AH90 polypeptide has the functions of repairing body surface wounds and accelerating scar recovery; meanwhile, the collagen also has certain skin repairing capacity. The fusion protein of AH90 polypeptide and collagen can better improve the biological activity, enhance the efficacy of repairing skin injury and repairing skin at lower onset concentration. The recombinant human collagen has good solubility, biocompatibility and safety, and has the functions of collagen and AH90 polypeptide; the preparation method of the recombinant human collagen has the advantages of low cost, high efficiency, mild production conditions, energy conservation and environmental protection, can realize industrial production, and has important application value in skin injury medicines.

Description

Recombinant human collagen and its preparation method and application

Technical Field

The present invention belongs to the technical field of recombinant proteins, and in particular relates to a recombinant human collagen protein and a preparation method and application thereof.

Background Art

Protein is the main bearer of human life activities, an important component of human tissue, and the main raw material for human tissue metabolism, renewal and repair. The role of protein in the human body also includes transporting substances, participating in the body's immunity, providing energy and regulating hormones. Among them, collagen is the most abundant protein in the human body, accounting for about 25-33% of the total protein in the body. According to statistics, collagen accounts for about 5% of the body weight, 75% of the skin weight, and 90% of the total volume of the skin, while collagen accounts for about 80% in connective tissue. Collagen exists in most tissues of the human body. Its function is to maintain the morphology and structure of the skin and tissues and organs, and it plays an indispensable role in the normal functioning of the human body.

Collagen is an extracellular protein, a fibrous protein twisted into a spiral shape by three peptide chains. Collagen contains a G-X-Y repeating sequence, also known as a collagen domain, where X/Y can be any amino acid except Gly, where X is often proline and Y is often hydroxyproline. Hydroxyproline residues can stabilize collagen molecules by forming intramolecular hydrogen bonds. The three α-peptide chains are wound into a triple helix structure in a parallel, right-handed spiral form through van der Waals forces, hydrogen bonds, and covalent cross-linking, so it has a very high tensile strength.

Peptides are a class of compounds formed by connecting multiple amino acids through peptide bonds. The connection method is the same as that of proteins. Usually, the number of amino acids in a polypeptide is less than 100. There are two main methods for synthesizing polypeptides: biosynthesis and chemical synthesis. Chemical synthesis is widely used for short peptides with ≤10 amino acids. Although chemical synthesis is mature and easy to industrialize, it also faces the disadvantage of sharply rising production costs for polypeptides with more than 10 amino acids. Such long peptides are often difficult to mass produce in industry. However, using the genetic recombination method in biosynthesis, polypeptides with more than 10 amino acids can be synthesized by Escherichia coli or other host cells.

However, in the process of expressing polypeptides in E. coli, the structure of the polypeptide leads to too fast biosynthesis rate and the formation of inclusion bodies, which increases the difficulty and cost of purification. Therefore, technicians often add protein tags such as SUMO and GST to enhance the solubility and expression level of the target product and improve the yield of the target product. The disadvantage is that additional proteases need to be added during the purification process to remove the protein tags.

AH90 polypeptide is one of the skin repair-promoting polypeptides of the stinking frog. It can be used to treat surface trauma, burns and skin ulcers. It can also reduce scar formation and accelerate scar repair. AH90 is a polypeptide with more than 10 amino acids, so one of its disadvantages is that it is difficult to synthesize and is expensive. There are two solutions: first, optimize the synthesis method to reduce the preparation cost of the AH90 polypeptide; second, without affecting the efficacy of the AH90 polypeptide, use new technology to reduce the actual usage of the AH90 polypeptide. This application uses fusion protein technology to successfully achieve the above goals.

CN112980865A discloses a method for constructing a recombinant human-like collagen engineering bacterium, based on the amino acid sequence of selected human-like collagen III, a gene sequence encoding recombinant human-like collagen is designed according to the preferred codons of Escherichia coli, and an enzyme digestion product is obtained by combining with the pET24a vector, and a human-like collagen fragment is obtained based on the enzyme digestion product, and the human-like collagen fragment is connected with the pET24a vector to obtain a PET24-human-like collagen fragment connection product, and finally a recombinant human-like collagen engineering bacterium is obtained based on the PET24-human-like collagen fragment connection product. The recombinant human-like collagen obtained by the inventive method has a certain inducing proliferation effect on human skin fibroblasts and human skin keratinocytes, but a single human-like collagen III cannot quickly repair skin damage.

CN102146135A discloses a recombinant human-like collagen protein, which has good hydrophilicity and stability, and its structure is 100% identical to the corresponding part of the natural collagen gene sequence. It will not cause immune rejection when applied to the human body, and can be widely used in the fields of biomedical materials, cosmetics, etc. The structure of the recombinant human-like collagen protein is a single-chain, single-helical structure, and its amino acids are 839, of which 1-241 are type III peptide segments, and 242-839 are type I peptide segments. However, the molecular weight of the recombinant human-like collagen protein is relatively large, and it has the disadvantage of being difficult to absorb when used in the fields of medicine and cosmetics.

At present, collagen is mainly extracted by traditional methods, but heterologous collagen may cause immune rejection when used, which lacks safety. In addition, strong acid, strong alkali and high temperature treatment are required during the extraction process, which increases the production cost and affects the activity of the product.

Therefore, how to provide a more gentle, more biologically active, safer and lower-cost method for preparing recombinant collagen has become an urgent problem to be solved.

Summary of the invention

In view of the deficiencies in the prior art and actual needs, the present invention provides a recombinant human collagen and a preparation method and application thereof. The recombinant human collagen has good biological activity, human compatibility and safety, is easy to prepare, has high production efficiency, can be used in the preparation of skin injury drugs and cosmetics, and can also be used for polypeptide display.

To achieve this object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a recombinant human collagen, wherein the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide;

The amino acid sequence of the collagen is shown in SEQ ID No.1.

Preferably, the amino acid sequence of the AH90 polypeptide is as shown in SEQ ID No.2.

Preferably, the amino acid sequence of the recombinant human collagen is as shown in SEQ ID No.3.

SEQ ID No.1 (amino acid sequence of collagen):

EAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGER.

SEQ ID No.2 (amino acid sequence of AH90 polypeptide):

ATAWDFGPHGLLPIRPIRIRPLCG.

SEQ ID No.3 (amino acid sequence of recombinant human collagen):

MATAWDFGPHGLLPIRPIRIRPLCGGGGGSEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERRTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFFCNMHHHHHH.

In SEQ ID No. 3, the amino acid sequence of the AH90 polypeptide is connected to the amino acid sequence of collagen through a peptide segment, and the amino acid sequence of the peptide segment is "GGGGS". The amino acid sequence of collagen is also connected to the C-PRO domain, which can enhance the structural stability of recombinant human collagen.

The sequence of the C-PRO domain is:

RTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNM.

In the present invention, the recombinant human collagen undergoes humanized transformation on the original collagen, thereby reducing immune rejection reactions and improving biocompatibility and safety; the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide, and the recombinant protein has stronger biological functions; the recombinant human collagen can be produced by genetic engineering, thereby reducing the contamination caused by pathogens such as microorganisms that may be brought by collagen from animal sources, and also avoiding the impact of the drastic methods in traditional extraction methods on the activity of collagen.

In the present invention, the AH90 polypeptide has the efficacy of repairing body surface trauma and accelerating scar repair. By connecting it with collagen, the efficacy of the recombinant protein in repairing skin damage can be further improved. It can be used as a raw material for skin damage drugs and cosmetics, and has practical application value.

In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant human collagen described in the first aspect.

Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No.4.

SEQ ID No.4 (nucleic acid molecule encoding recombinant human collagen):

atggctaccgcttgggacttcggtccgcacggtctgctgccgatccgtccgatccgtatccgtccgctgtgcggtggtggtggtggttctgaagctggtctgccgggtgctaaaggtctgaccggttctccgggttctccgggtccggacggtaaaaccggtccgccgggtccggctggtcaggacggtc gtccgggtccgccgggtccgccgggtgctcgtggtcaggctggtgttatgggtttcccgggtccgaaaggtgc tgctggtgaaccgggtaaagctggtgaacgtggtgttccgggtccgccgggtgctgttggtccggctggtaaagacggtgaagctggtgctcagggtccgccgggtccggctggtccggctggtgaacgtcgtacctgccgtgacctgaaaaatgtgccactctgactggaaatctggtgaatactggatcgac ccgaaccagggttgcaacttagacgctataaaagttttctgcaacatgcaccaccaccaccaccactaa.

In the present invention, the coding sequence encoding the recombinant human collagen is humanized and codon optimized, thereby increasing the safety of the recombinant protein, improving the production efficiency, and promoting the promotion and use of related products.

In a third aspect, the present invention provides an expression vector comprising at least one copy of the nucleic acid molecule described in the second aspect.

In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the recombinant human collagen described in the first aspect.

Preferably, the nucleic acid molecule described in the second aspect is integrated into the genome of the recombinant cell, or the recombinant cell contains the expression vector described in the third aspect.

In a fifth aspect, the present invention provides a method for preparing the recombinant human collagen described in the first aspect, the preparation method comprising:

(1) connecting the nucleic acid molecule encoding the recombinant human collagen into a plasmid vector to construct an expression vector, transferring the expression vector into a recipient cell, and screening to obtain a positive clone;

(2) culturing the positive clone obtained in step (1), adding an inducer to the fermentation broth for induction, and collecting the bacterial cells after induction of expression;

(3) crushing the bacterial cells obtained in step (2) and collecting the supernatant by centrifugation;

(4) Purifying the supernatant obtained in step (3) to obtain the recombinant human collagen.

In the present invention, the recombinant protein is prepared by genetic engineering, the product has better safety and biological activity, and the production efficiency is improved, creating conditions for the factory production of the recombinant protein.

Preferably, in step (1), the recipient cell comprises an Escherichia coli expression strain.

Preferably, in step (2), the induction treatment method comprises induction using IPTG.

Preferably, the final concentration of IPTG used for IPTG induction is 0.5-1.5 mM, for example, it can be 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM.

Preferably, the OD value of the induction treatment is 0.6-0.8, for example, 0.6, 0.7 or 0.8.

Preferably, the temperature of the induction treatment is 36-38°C, for example, it can be 36°C, 37°C or 38°C, the rotation speed of the induction treatment is 120-320rpm, for example, it can be 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, 220rpm, 240rpm, 260rpm, 280rpm, 300rpm or 320rpm, etc., and the time of the induction treatment is 4-6h, for example, it can be 4h, 5h or 6h, etc.

Preferably, in step (3), the bacterial cells are disrupted in a manner comprising the following steps: resuspending the bacterial cells with PB solution, performing high-pressure homogenization, collecting the supernatant liquid by centrifugation and filtering.

Preferably, the mass ratio of the bacteria to the PB solution is 1:(5-50), for example, it can be 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50, etc.

Preferably, the molar concentration of the PB solution is 10-30 mM, for example, it can be 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM or 30 mM.

Preferably, the high-pressure homogenization step is: breaking the bacteria 2 to 4 times, for example 2 times, 3 times or 4 times, under the condition of 600 to 1000 bar (for example, 600 bar, 700 bar, 800 bar, 900 bar or 1000 bar, etc.).

Preferably, the centrifugal rotation speed is 12000-16000 rpm (for example, it can be 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm or 16000 rpm, etc.), and the centrifugal time is 40-60 min (for example, it can be 40 min, 45 min, 50 min, 55 min or 60 min, etc.).

Preferably, the pore size of the filter membrane used for the filtration is 0.2-0.8 μm, for example, it can be 0.22 μm, 0.4 μm, 0.45 μm, 0.5 μm or 0.8 μm.

Preferably, in step (4), the purification method comprises: using a nickel ion column for adsorption purification, sequentially using a washing buffer and an elution buffer for elution, and then using a phenyl column to purify the eluate.

Preferably, the washing buffer is a 10-30 mM PB solution, for example, it can be 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM or 30 mM, etc.

Preferably, the elution buffer is a 10-30mM Tris solution, for example, it can be 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM or 30mM, etc.

Preferably, the elution buffer used for the purification is a 10-30mM Tris solution, for example, it can be 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM or 30mM, etc.

As a preferred technical solution, the method for preparing recombinant human collagen of the present invention comprises the following steps:

(1) connecting the nucleic acid molecule encoding the recombinant human collagen into a plasmid vector to construct an expression vector, and transferring the expression vector into an Escherichia coli expression strain to screen and obtain positive clones;

(2) culturing the positive clone obtained in step (1), adding IPTG with a final concentration of 0.5 to 1.5 mM to the fermentation broth to induce protein expression, wherein the OD value of the induction treatment is 0.6 to 0.8, the induction treatment temperature is 36 to 38° C., the induction treatment speed is 120 to 320 rpm, the induction treatment time is 4 to 6 hours, and the bacteria are collected after induction expression;

(3) resuspending the cells with a PB solution having a molar concentration of 10 to 30 mM, wherein the mass ratio of the cells to the PB solution is 1:(5 to 50), performing high-pressure homogenization, breaking the cells 2 to 4 times at 600 to 1000 bar, centrifuging at 12000 to 16000 rpm for 40 to 60 min, collecting the supernatant, and filtering with a filter membrane having a pore size of 0.2 to 0.8 μm;

(4) Using a nickel ion column to adsorb and purify the supernatant obtained in step (3), and eluting with a washing buffer and an elution buffer in sequence, wherein the washing buffer is a 10-30 mM PB solution, and the elution buffer is a 10-30 mM Tris solution; and then using a phenyl column to purify the eluate, wherein the elution buffer used for the purification is a 10-30 mM Tris solution, to obtain the recombinant human collagen.

In a sixth aspect, the present invention provides the use of the recombinant human collagen described in the first aspect in polypeptide display.

In the present invention, the polypeptide can be connected to the N-terminus or C-terminus of the recombinant human collagen in series, and the collagen fragment and the active polypeptide can be co-expressed by gene recombination. Since collagen has strong solubility, the collagen fragment carrying the active polypeptide can also be efficiently expressed in Escherichia coli. The types of active polypeptides are not limited to the amino acid sequence of the AH90 polypeptide, but are also applicable to other active polypeptides.

In a seventh aspect, the present invention provides use of the recombinant human collagen described in the first aspect in the preparation of medicines or cosmetics for treating skin damage.

In an eighth aspect, the present invention provides a composition for treating skin damage and repairing skin, wherein the composition for treating skin damage and repairing skin contains the recombinant human collagen described in the first aspect.

Preferably, the composition for treating skin damage and repairing the skin comprises, by weight: 0.05-0.5 parts of recombinant human collagen, 0.05-0.5 parts of blue copper peptide, 2-5 parts of white jelly gum, 0.5-1.5 parts of silk protein and 90-100 parts of water.

In the present invention, the recombinant human collagen is 0.05 to 0.5 parts by weight, for example, it can be 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 parts; the blue copper peptide is 0.05 to 0.5 parts by weight, for example, it can be 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 parts. 5 parts, etc.; the white ji glue is counted in parts by weight at 2-5 parts, for example, it can be 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts or 5 parts, etc.; the silk protein is counted in parts by weight at 0.5-1.5 parts, for example, it can be 0.5 parts, 0.6 parts, 0.8 parts, 1 parts, 1.2 parts, 1.4 parts or 1.5 parts, etc.; the water is counted in parts by weight at 90-100 parts, for example, it can be 90 parts, 91 parts, 92 parts, 93 parts, 94 parts, 95 parts, 96 parts, 97 parts, 98 parts, 99 parts or 100 parts, etc.

The silk protein and white ji glue in the composition are both moisturizing and water-locking ingredients, which can increase the moisture content of the skin and repair the skin barrier; blue copper peptide can stimulate the production of glycosaminoglycans, a cell matrix component, and can also indirectly stimulate collagen production, promoting the skin's repair ability. Recombinant human collagen (AH90-ColI) can directly supplement the body's collagen and stimulate the integrin receptors of cells, thereby directly stimulating cell proliferation and migration to achieve a repair effect. The two have a synergistic effect.

Compared with the prior art, the present invention has the following gain effects:

(1) The present invention utilizes a gene recombination method to connect collagen fragments and AH90 polypeptides in series. The product has the functions of both collagen and AH90 polypeptides, and has higher biological activity. It can effectively repair various types of skin damage and has strong practical value. In addition, the protein fragment used in the present invention is human collagen, which reduces immune rejection and greatly improves human compatibility and safety. The preparation method of the recombinant protein of the present invention is free of microbial contamination from pathogens from animals, and avoids the use of strong acid, strong alkali, high temperature and other drastic means during the production process, thereby ensuring the activity and safety of the recombinant protein.

(2) The recombinant protein in the present invention is prepared by expression and purification in E. coli. The target product exists in a soluble form in E. coli with high yield, and no enzyme cleavage step is required after the product is purified. It is suitable for displaying polypeptides of various lengths, especially for polypeptides with amino acid numbers greater than 10. This method can effectively reduce production costs and improve production efficiency.

(3) A composition for treating skin damage according to the present invention, wherein the collagen fused with the AH90 polypeptide is human collagen. Since human collagen has better compatibility with the human body, the fusion protein containing the AH90 polypeptide fragment is more easily absorbed by the human body, which reduces the minimum effective amount of the AH90 polypeptide. Therefore, the actual amount of the AH90 polypeptide used can be reduced while ensuring the same or better efficacy, thereby solving the problem of high cost of the AH90 polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the pET28a(+) plasmid map in Example 1;

FIG. 2 is a polyacrylamide gel electrophoresis diagram of AH90-ColI in Example 1.

DETAILED DESCRIPTION

To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

The sources of the experimental materials used in the following examples and comparative examples are shown in Table 1:

Table 1

Components factory Part Number pET28a vector Suzhou Hongxun Biotechnology Co., Ltd. / BL21(DE3) cells Suzhou Hongxun Biotechnology Co., Ltd. / Kanamycin Shanghai Bioengineering Co., Ltd. KK90558- IPTG Shanghai Bioengineering Co., Ltd. KI90542 NI column Changzhou Tiandirenhe Biotechnology Co., Ltd. SA052100 Phenyl HS Column Changzhou Tiandirenhe Biotechnology Co., Ltd. SH006100 Commercially available collagen Ctrl-Col Guangzhou Yousheng Biotechnology Co., Ltd. / Blue Copper Peptide Shenzhen Dickman Biotechnology Co., Ltd. / White gum Zefang (Guangzhou) Biological Co., Ltd. / Silk protein Zefang (Guangzhou) Biological Co., Ltd. /

Example 1

In this embodiment, a recombinant human collagen is prepared. The recombinant human collagen is a fusion protein of collagen and AH90 polypeptide (referred to as AH90-ColI collagen). The amino acid sequence of the recombinant human collagen is shown in SEQ ID No.3.

The recombinant human collagen is prepared by the following method:

1. Host construction

The collagen fragment (amino acid sequence as shown in SEQ ID No. 1) and the AH90 polypeptide sequence (amino acid sequence as shown in SEQ ID No. 2) were concatenated in a 1:1 manner, and a 6×His tag was added to the C-terminus. The nucleic acid molecule sequence encoding the protein (nucleotide sequence as shown in SEQ ID No. 4) was constructed into the pET28a vector (purchased from Suzhou Hongxun Biotechnology Co., Ltd.) after E. coli codon optimization. The pET28a (+) plasmid map is shown in Figure 1. The constructed expression vector was introduced into the E. coli expression strain BL21 (DE3) cells (purchased from Suzhou Hongxun Biotechnology Co., Ltd.).

2. Bacterial expression

A single clone of recombinant E. coli was picked and transferred to 50 mL LB medium, and kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 50 μg/mL was added, and cultured overnight at 37°C and 220 rpm. On the second day, the bacterial solution was aspirated from the seed solution and inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C and 220 rpm. When the OD value was in the range of 0.6 to 0.8, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 1 mM was added to induce protein expression. The induction treatment temperature was 37°C, the induction treatment speed was 220 rpm, and the bacteria were collected by centrifugation after 5 hours.

3. Bacteria disruption

Take the collected bacteria, add 20mM PB solution at a mass ratio of 1:10 to resuspend the bacteria, use a high-pressure homogenizer at 800bar to break the bacteria for 3 times until the bacterial solution is clear and transparent, and the broken bacteria can be observed under a microscope; centrifuge the bacterial solution at 14000rpm for 50min, collect the supernatant, and filter it using a 0.45μm filter membrane.

4. Purification

The filtered supernatant was adsorbed on a nickel ion column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), washed with a 20 mM Pb solution, and then eluted with a 20 mM Tris solution. The eluate was further purified using a phenyl column (Phenyl HS column) (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and the target protein was eluted with a 20 mM Tris solution to obtain the recombinant human collagen.

5. Protein Identification and Preservation

Take an appropriate amount of protein solution for SDS-PAGE detection, and use the grayscale scanning method to determine the proportion of the recombinant human collagen in the entire lane, which is the purity; the polyacrylamide gel electrophoresis diagram of AH90-ColI is shown in Figure 2, in which Lane 3 to 5 shows the actual molecular weight of the recombinant collagen, which is about 23 kDa. The purity of the purified recombinant collagen can reach 90%; the remaining protein treatment solution is stored at low temperature (-20 to -80°C) for a long time or the protein is freeze-dried into powder.

Example 2

In this embodiment, a recombinant human collagen is prepared, and the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide, and the amino acid sequence of the recombinant human collagen is shown in SEQ ID No. 3. The recombinant human collagen is prepared by the following method:

1. Host construction

Same as Example 1.

2. Bacterial expression

A single clone of recombinant E. coli was picked and transferred to 50 mL LB medium, and kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 40 μg/mL was added, and cultured overnight at 36°C and 170 rpm. On the second day, the bacterial solution was aspirated from the seed solution and inoculated into fresh LB medium at a ratio of 1:100, and cultured at 36°C and 170 rpm. When the OD value was in the range of 0.6 to 0.8, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 0.8 mM was added to induce protein expression. The induction treatment temperature was 36°C, the induction treatment speed was 170 rpm, and the bacteria were collected by centrifugation after 6 hours.

3. Bacteria disruption

Take the collected bacteria, add 15mM PB solution at a mass ratio of 1:10 to resuspend the bacteria, use a high-pressure homogenizer at 700bar to break the bacteria for 3 times until the bacterial solution is clear and transparent, and the broken bacteria can be observed under a microscope; centrifuge the bacterial solution at 13000rpm for 60min, collect the supernatant, and filter it using a 0.45μm filter membrane.

4. Purification

The filtered supernatant was adsorbed on a nickel ion column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), washed with a 15mM Pb solution, and then eluted with a 15mM Tris solution. The eluate was further purified with a Phenyl HS column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and finally the target protein was eluted with a 15mM Tris solution to obtain the recombinant human collagen.

5. Protein Identification and Preservation

Same as Example 1.

Example 3

In this embodiment, a recombinant human collagen is prepared, and the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide, and the amino acid sequence of the recombinant human collagen is shown in SEQ ID No. 3. The recombinant human collagen is prepared by the following method:

1. Host construction

Same as Example 1.

2. Bacterial expression

A single clone of recombinant E. coli was picked and transferred to 50 mL LB medium, and kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 60 μg/mL was added, and cultured overnight at 38°C and 270 rpm. On the second day, the bacterial solution was aspirated from the seed solution and inoculated into fresh LB medium at a ratio of 1:100, and cultured at 38°C and 270 rpm. When the OD value was in the range of 0.6 to 0.8, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 1.2 mM was added to induce protein expression. The induction treatment temperature was 38°C, the induction treatment speed was 270 rpm, and the bacteria were collected by centrifugation after 4 hours.

3. Bacteria disruption

Take the collected bacteria, add 25mM PB solution at a mass ratio of 1:10 to resuspend the bacteria, use a high-pressure homogenizer at 900bar to break the bacteria for 3 times until the bacterial solution is clear and transparent, and the broken bacteria can be observed under a microscope; centrifuge the bacterial solution at 15000rpm for 40min, collect the supernatant, and filter it using a 0.45μm filter membrane.

4. Purification

The filtered supernatant was adsorbed on a nickel ion column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), first washed with a 25mM Pb solution, and then eluted with a 25mM Tris solution. The eluate was further purified with a Phenyl HS column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and finally the target protein was eluted with a 25mM Tris solution to obtain the recombinant human collagen.

5. Protein Identification and Preservation

Same as Example 1.

Example 4

In this embodiment, a recombinant human collagen is prepared, and the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide, and the amino acid sequence of the recombinant human collagen is shown in SEQ ID No. 3. The recombinant human collagen is prepared by the following method:

1. Host construction

Same as Example 1.

2. Bacterial expression

A single clone of recombinant E. coli was picked and transferred to 50 mL LB medium, and kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 50 μg/mL was added, and cultured overnight at 37°C and 120 rpm. On the second day, the bacterial solution was aspirated from the seed solution and inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C and 120 rpm. When the OD value was in the range of 0.6 to 0.8, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 0.5 mM was added to induce protein expression. The induction treatment temperature was 37°C, the induction treatment speed was 120 rpm, and the bacteria were collected by centrifugation after 5 hours.

3. Bacteria disruption

Take the collected bacteria, add 10mM PB solution at a mass ratio of 1:10 to resuspend the bacteria, use a high-pressure homogenizer at 600bar to break the bacteria for 3 times until the bacterial solution is clear and transparent, and the broken bacteria can be observed under a microscope; centrifuge the bacterial solution at 16000rpm for 40min, collect the supernatant, and filter it using a 0.45μm filter membrane.

4. Purification

The filtered supernatant was adsorbed on a nickel ion column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), washed with a 10 mM Pb solution, and then eluted with a 10 mM Tris solution. The eluate was further purified using a phenyl column (Phenyl HS column) (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and finally the target protein was eluted with a 10 mM Tris solution to obtain the recombinant human collagen.

5. Protein Identification and Preservation

Same as Example 1.

Example 5

In this embodiment, a recombinant human collagen is prepared, and the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide, and the amino acid sequence of the recombinant human collagen is shown in SEQ ID No. 3. The recombinant human collagen is prepared by the following method:

1. Host construction

Same as Example 1.

2. Bacterial expression

A single clone of recombinant E. coli was picked and transferred to 50 mL LB medium, and kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 50 μg/mL was added, and cultured overnight at 37°C and 320 rpm. On the second day, the bacterial solution was aspirated from the seed solution and inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C and 320 rpm. When the OD value was in the range of 0.6 to 0.8, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd.) with a final concentration of 1.5 mM was added to induce protein expression. The induction temperature was 37°C, the induction speed was 320 rpm, and the bacteria were collected by centrifugation after 5 hours.

3. Bacteria disruption

Take the collected bacteria, add 30mM PB solution at a mass ratio of 1:10 to resuspend the bacteria, use a high-pressure homogenizer at 1000bar to break the bacteria for 3 times until the bacterial solution is clear and transparent, and the broken bacteria can be observed under a microscope; centrifuge the bacterial solution at 12000rpm for 60min, collect the supernatant, and filter it using a 0.45μm filter membrane.

4. Purification

The filtered supernatant was adsorbed on a nickel ion column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), washed with a 30 mM Pb solution, and then eluted with a 30 mM Tris solution. The eluate was further purified using a phenyl column (Phenyl HS column) (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and finally the target protein was eluted with a 30 mM Tris solution to obtain the recombinant human collagen.

5. Protein Identification and Preservation

Same as Example 1.

Example 6

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, the temperature for inducing expression is 35°C, and the remaining steps refer to Example 1.

Example 7

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, the temperature for inducing expression is 39° C., and the remaining steps refer to Example 1.

Example 8

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, the rotation speed for inducing expression is 100 rpm, and the remaining steps refer to Example 1.

Example 9

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, the rotation speed for inducing expression is 340 rpm, and the remaining steps refer to Example 1.

Example 10

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, in the bacterial cell crushing step, the concentration of the PB solution used for resuspending the bacteria is 5 mM, and the remaining steps refer to Example 1.

Embodiment 11

In this example, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this example and Example 1 is that in the preparation method of the recombinant human collagen, in the bacterial cell crushing step, the concentration of the PB solution used for resuspending the bacteria is 35 mM, and the remaining steps refer to Example 1.

Example 12

In this example, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this example and Example 1 is that in the preparation method of the recombinant human collagen, in the nickel ion column purification step, the concentration of the PB solution used for elution is 5 mM, and the remaining steps refer to Example 1.

Example 13

In this example, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this example and Example 1 is that in the preparation method of the recombinant human collagen, in the nickel ion column purification step, the concentration of the PB solution used for elution is 35 mM, and the remaining steps refer to Example 1.

Embodiment 14

In this example, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this example and Example 1 is that in the preparation method of the recombinant human collagen, in the nickel ion column purification step, the concentration of the Tris solution used for elution is 5 mM, and the remaining steps refer to Example 1.

Embodiment 15

In this example, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this example and Example 1 is that in the preparation method of the recombinant human collagen, in the nickel ion column purification step, the concentration of the Tris solution used for elution is 35 mM, and the remaining steps refer to Example 1.

Example 16

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, in the phenyl column purification step, the concentration of the Tris solution used for elution is 5 mM, and the remaining steps refer to Example 1.

Embodiment 17

In this embodiment, a recombinant human collagen is prepared. The amino acid sequence of the recombinant human collagen is consistent with that in Example 1. The only difference between this embodiment and Example 1 is that in the preparation method of the recombinant human collagen, in the phenyl column purification step, the concentration of the Tris solution used for elution is 35 mM, and the remaining steps refer to Example 1.

The recombinant human collagen prepared by the preparation methods in Examples 1 to 17 was tested for purity. The test results are shown in Table 2.

Table 2

It can be seen from Table 2 that the purity of the recombinant human collagen obtained by the preparation methods in Examples 1 to 5 is 89.2% to 92.6%, and the yield of the recombinant human collagen obtained is high, which can save costs.

Comparative Example 1

This comparative example provides a human collagen protein ColI, the amino acid sequence of which is shown in SEQ ID NO.1.

Comparative Example 2

This comparative example provides a commercially available collagen protein Ctrl-Col, which is purchased from a biotechnology company in Jiangsu, and whose amino acid sequence is different from SEQ ID NO.1.

Comparative Example 3

This comparative example provides an AH90 polypeptide, the amino acid sequence of the AH90 polypeptide is shown in SEQ ID NO.2.

Comparative Example 4

This comparative example provides a fingerless frog polypeptide (RGP), which is purchased from Shanghai Newpu Biotechnology Co., Ltd., and the amino acid sequence is shown in SEQ ID NO.5.

SEQ ID NO. 5: GIFGKILGVGKKVLCGLSGMC.

Comparative Example 5

This comparative example provides a collagen-RGP fusion protein (RGP-ColI), the amino acid sequence of the fingerless disc frog polypeptide (RGP) is shown in SEQ ID NO.5, and the amino acid sequence of the collagen (ColI) is shown in SEQ ID NO.1. The difference between this comparative example and Example 1 is that the fingerless disc frog polypeptide (RGP) replaces the AH90 polypeptide in the fusion protein of Example 1; the preparation method of the collagen-RGP fusion protein (RGP-ColI) refers to Example 1.

Comparative Example 6

This comparative example provides a mixture of collagen ColI and AH90, wherein the amino acid sequence of the collagen ColI is shown as SEQ ID NO.1, and the amino acid sequence of AH90 is shown as SEQ ID NO.2.

Application Example 1

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen (AH90-ColI) described in Example 1, and the composition comprises, by weight: 0.1 parts of recombinant human collagen, 0.1 parts of blue copper peptide, 3 parts of white jelly gum, 1 part of silk protein and 95.8 parts of water.

The preparation method of the composition is as follows: recombinant human collagen, blue copper peptide, white schizonepeta gelatin, silk protein and water are mixed in sequence to obtain the composition for treating skin damage.

Application Example 2

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0.25 parts of recombinant human collagen, 0.3 parts of blue copper peptide, 4 parts of white jelly gum, 1.2 parts of silk protein and 94.25 parts of water.

The preparation method of the composition refers to Application Example 1.

Application Example 3

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0.75 parts of recombinant human collagen, 0.8 parts of blue copper peptide, 2.5 parts of white jelly gum, 0.8 parts of silk protein and 95.15 parts of water.

The preparation method of the composition refers to Application Example 1.

Application Example 4

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0.05 parts of recombinant human collagen, 0.5 parts of blue copper peptide, 5 parts of white jelly gum, 0.5 parts of silk protein and 93.95 parts of water.

The preparation method of the composition refers to Application Example 1.

Application Example 5

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0.5 parts of recombinant human collagen, 0.05 parts of blue copper peptide, 2 parts of white jelly gum, 1.5 parts of silk protein and 95.95 parts of water.

The preparation method of the composition refers to Application Example 1.

Application Example 6

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0.2 parts of recombinant human collagen, 0 parts of blue copper peptide, 3 parts of white jelly gum, 1 part of silk protein and 95.8 parts of water.

The preparation method of the composition refers to Application Example 1.

Application Example 7

This application example provides a composition for treating skin damage and repairing the skin, wherein the composition contains the recombinant human collagen described in Example 1, and the composition comprises, by weight: 0 parts of recombinant human collagen, 0.2 parts of blue copper peptide, 3 parts of white jelly gum, 1 part of silk protein and 95.8 parts of water.

The preparation method of the composition refers to Application Example 1.

Comparative application example 1

This comparative application example provides a composition with skin care effects. The difference between the composition and application example 1 is that the collagen ColI provided in comparative example 1 replaces the AH90-ColI in application example 1.

Comparative Application Example 2

This comparative application example provides a composition with skin care effects. The difference between the composition and application example 1 is that the collagen Ctrl-Col provided in comparative example 2 replaces the AH90-ColI fusion protein in application example 1.

Comparative Application Example 3

This comparative application example provides a composition with skin care effects. The difference between the composition and application example 1 is that the AH90 polypeptide provided in comparative example 3 replaces the AH90-ColI in application example 1.

Comparative Application Example 4

This comparative application example provides a composition with skin care effect. The difference between the composition and application example 1 is that the fingerless frog polypeptide (RGP) provided in comparative example 4 replaces the AH90-ColI fusion protein in application example 1.

Comparative Application Example 5

This comparative application example provides a composition with skin care effect. The difference between the composition and application example 1 is that the RGP-ColI fusion protein provided in comparative example 5 replaces the AH90-ColI fusion protein in application example 1.

Comparative Application Example 6

This comparative application example provides a composition with skin care effects, and the difference between the composition and application example 1 is that the mixture of collagen (ColI) and AH90 polypeptide provided in comparative example 6 replaces the AH90-ColI fusion protein in application example 1.

Test Example 1: 3T3 cell proliferation detection

BALB/3T3 cells are mouse fibroblasts and are a common cell model for evaluating skin efficacy. As the main cells that make up the dermis of the skin, BALB/3T3 cells are closely related to skin aging and damage. Promoting their proliferation has a positive effect on skin anti-aging and repair.

In the process of wound repair, fibroblasts have the function of synthesizing and secreting a large amount of collagen fibers and matrix components, forming granulation tissue together with new capillaries, filling wound tissue defects and creating conditions for epidermal cell coverage. Therefore, the proliferation experiment of BALB/3T3 fibroblasts is also often used to measure the degree of wound healing and skin repair.

3T3 cells with good growth status were plated in 96-well plates. After culturing for 24 hours, the culture medium was discarded and DMEM basal culture medium containing different samples was added. Three replicate wells were set for each sample. After 48 hours, the OD ₄₅₀ and OD ₆₃₀ values were detected by CCK8 method to obtain the effect of the sample on the proliferation of 3T3 cells.

The calculation formula of relative cell viability is:

Table 3

Samples to be tested Relative Vitality Samples to be tested Relative Vitality Blank control 100% Comparative Example 1 112% Positive Control 152% Comparative Example 2 110% Example 1 136% Comparative Example 3 111% Example 2 133% Comparative Example 4 109% Example 3 134% Comparative Example 5 123% Example 4 129% Comparative Example 6 128% Example 5 131% / /

The test results are shown in Table 3. The AH90-ColI collagen of Examples 1 to 5 can promote the growth of 3T3 cells, and there is a significant statistical difference compared with the blank group (p<0.05).

Test Example 2: Zebrafish collagen detection

Skin growth, repair, elasticity and tension are all related to collagen, and its loss will reduce skin smoothness and produce wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two α peptide chains and one α2 chain, encoded by col1a1a and col1a2 genes, respectively, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, namely colIa1a, col1a1b and col1a2, which encode α1(I), α2(I) and α3(I) chains. Therefore, by detecting the relative expression of colIa1a, col1a1b or col1a2 genes in different samples, it can be indicated whether the sample has anti-wrinkle and repair effects.

Experimental method: The highest concentration of the experiment shall not be higher than the NOEC value determined in the preliminary experiment. According to the experimental requirements, the concentration of the test substance shall be diluted in equal proportions.

According to the experimental requirements, a sufficient number of normally developed 96hpf zebrafish fry were pre-screened and randomly distributed into 6-well plates, 30 per well, and 3 wells per concentration group. The buffer water in the 6-well plate was removed without harming the fry, and 3 mL of the test solution of different concentrations was added. The blank control group was added with buffer water. The culture plate was covered and incubated in a biochemical incubator at 28.5℃±0.5℃ for 24h.

According to the instructions of the RNA rapid extraction kit, the total RNA of each group of zebrafish was extracted, and the total RNA was measured using an ultra-micro UV spectrophotometer, requiring the absorbance ratio of each experimental group at 260nm and 280nm to be between 1.90-2.20. The extracted RNA was synthesized into cDNA according to the instructions of the reverse transcription kit for later use.

The SYBR Green fluorescent dye kit was used to spot the plate and configure a 20μL reaction system. It contains 10μL SYBRGreen Mix, 1μL upstream and downstream primers, 2μL DNA template, and 7μL DEPC water. The reaction solution was added to the reaction wells, and then the PCR reaction program was run on the fluorescent quantitative PCR instrument. The specific operating parameters are as follows: 94℃, 30s; 94℃, 5s, 61℃, 35s; 98℃, 10s; 65℃, 60s; 98℃, 1s; 40 cycles.

The Ct values of qPCR detection of the internal reference gene (β-actin) and target gene col1a1a in each group were recorded.

The test results of zebrafish collagen Col1a1a expression are shown in Table 4:

Table 4

Samples to be tested Relative expression of Col1a1a Samples to be tested Relative expression of Col1a1a Blank control 100% Comparative Example 1 110% Example 1 129% Comparative Example 2 106% Example 2 125% Comparative Example 3 108% Example 3 127% Comparative Example 4 107% Example 4 120% Comparative Example 5 116% Example 5 121% Comparative Example 6 117%

From the results in Table 4, it can be seen that the collagen-AH90 fusion protein provided by Examples 1-5 can significantly increase the relative expression level of the collagen col1a1a gene compared with the blank control group and other comparative examples, and has the effect of repairing the skin.

Test case 3: Security assessment

30 volunteers who meet the requirements, aged 18-60 years old, 22 females and 8 males, were selected to test the fusion proteins obtained in application examples 1-7 and the compositions obtained in comparative application examples 1-6. 0.025 mL of the test sample solution was taken respectively and placed in the small chamber of the spot tester, and 10 test points were selected in each of the left and right arms of the subject. The spot tester with the test sample was applied to the inner side of the subject's forearm with a low-allergenic tape, and lightly pressed with the palm of the hand to evenly apply it to the skin, and waited for 24 hours. After removing the spot tester for 30 minutes, 24 hours, and 48 hours (after the indentation disappears), observe the skin reaction according to the standards in Table 5.

Table 5

Table 6

The experimental results of the safety evaluation are shown in Table 6. The results show that application examples 1-7 are all negative reactions, indicating that the safety of the composition and its derivative products of the present invention is guaranteed, and there are no adverse reactions such as skin irritation and sensitization (except for people who are prone to allergies or people who are allergic to this product).

Test Example 4: Skin Repair Test

39 volunteers were recruited, aged between 18 and 60, including 29 females and 10 males, to test the repairing effects of Application Examples 1-7 and Comparative Application Examples 1-6, respectively. Each sample was tested on 3 subjects, and each person was tested three times. Three experimental sites were selected on the inner side of the subjects' arms, and 0.5% sodium dodecyl sulfate (SLS) solution was repeatedly applied to the skin to form a chemical damage model. Then, 50 μg/mL of the cream prepared in Application Examples 1-7 and Comparative Application Examples 1-6 was applied to the damaged skin, once in the morning and once in the evening. After continuous application for 14 days and 28 days, the changes in the transepidermal water loss value (TEWL) of the skin were recorded. The results are shown in Table 7.

The calculation formula of the rate of change is:

Change rate = (TEWL _{(day n)} -TEWL _{(day 0)} ) / TEWL _{(day 0)} × 100%

Table 7

The results in Table 7 show that under natural conditions, the skin barrier of the acute injury model will be naturally repaired, and the transepidermal water loss value will gradually decrease. The use of the AH90-ColI recombinant human collagen provided by the present invention will accelerate the repair process of the skin barrier.

It can be seen from Application Examples 1, 6 and 7 that the AH90-ColI recombinant human collagen and the blue copper peptide in the composition produce a synergistic effect, which is better than the effect of AH90-ColI recombinant human collagen (Application Example 6) or blue copper peptide (Application Example 7) alone.

By comparing Application Example 1 (AH90-ColI) with Comparative Application Example 6 (a mixture of AH90 and ColI), it can be seen that the repair effect of Application Example 1 after 14 days and 28 days of use is better than that of the ordinary mixture of Comparative Application Example 6, indicating that AH90-ColI recombinant human collagen is more effective than the mixture of AH90 polypeptide and collagen ColI.

Application Example 1 is the most preferred embodiment of the present invention. Under the premise of ensuring the efficacy of wound repair and skin repair, the amount of AH90-ColI recombinant human collagen used is only 0.1%, and the actual amount of AH90 polypeptide used is very low. This is because human collagen has good compatibility with the human body, so connecting human collagen with AH90 polypeptide can make AH90 polypeptide more easily absorbed, reduce the minimum effective amount of AH90, thereby reducing the actual amount of AH90 polypeptide used, and further reducing the cost of AH90 polypeptide products.

Test Example 5: Skin redness test

Test instrument: Skin color test probe Colorimeter CL400.

A total of 39 subjects, aged between 18 and 60, including 29 females and 10 males, were tested on the compositions provided in Application Examples 1 to 7 and Comparative Application Examples 1 to 6. The subjects' faces were first rinsed with clean water and then dried with a non-chip absorbent paper towel. The subjects then sat quietly in a standard environment (temperature 20° C. to 22° C., humidity 40% to 60%) for 30 minutes.

Test method: Before using the composition provided in Application Examples 1-7 and Comparative Application Examples 1-6, the face was selected as the test area, and the skin redness value (a value) of the test area was measured. Each test site was measured 3 times, and the average value was taken and recorded as W0; the subjects returned to the laboratory after using the composition provided in Application Examples 1-7 and Comparative Application Examples 1-6 every day for 14 days and 28 days, and the skin redness value (a value) of the test area was measured, recorded as W2 and W4. The test results are shown in Table 8.

The calculation formula of the rate of change is:

W2 skin redness value change rate = (W2-W0)/W0×100%

W4 skin redness value change rate = (W4-W0)/W0×100%

Table 8

The skin red value (a value) represents the amount of hemoglobin or erythema (indicating the change in color from green to red). The lower the value, the lower the hemoglobin or erythema. As can be seen from Table 8, the skin red value of Application Example 1 decreases faster than that of Comparative Application Example 6, which indicates that the AH90-ColI recombinant protein provided in Application Example 1 has a better effect on wound repair and skin repair than the mixture of AH90 polypeptide and collagen in Comparative Application Example 6.

In summary, the AH90-ColI recombinant human collagen prepared by the present invention has the functions of both collagen and AH90 polypeptide, has good solubility and high biocompatibility; it avoids the pathogen contamination that may exist in the traditional collagen extraction process, and has higher safety; the preparation method is mature in technology, avoids the use of drastic means such as strong acid, strong alkali and high temperature, has better biological activity, and has high production efficiency and low cost, and can realize factory production, which has important application significance in the preparation of skin injury treatment drugs and cosmetics.

The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (10)

1. The recombinant human collagen is characterized in that the recombinant human collagen is a fusion protein of collagen and AH90 polypeptide;

the amino acid sequence of the collagen is shown as SEQ ID No. 1.

2. The recombinant human collagen according to claim 1, wherein the amino acid sequence of said AH90 polypeptide is shown in SEQ ID No. 2;

preferably, the amino acid sequence of the recombinant human collagen is shown as SEQ ID No. 3.

3. A nucleic acid molecule encoding the recombinant human collagen of claim 1 or 2;

preferably, the nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No. 4.

4. An expression vector comprising at least one copy of the nucleic acid molecule of claim 3.

5. A recombinant cell expressing the recombinant human collagen of claim 1 or 2;

preferably, the recombinant cell has the nucleic acid molecule of claim 3 integrated into its genome or contains the expression vector of claim 4.

6. The method for preparing recombinant human collagen according to claim 1 or 2, comprising:

(1) Connecting the nucleic acid molecule for encoding the recombinant human collagen into a plasmid vector to construct an expression vector, transferring the expression vector into a receptor cell, and screening to obtain a positive clone;

(2) Culturing the positive clone obtained in the step (1), adding an inducer into fermentation liquor for induction treatment, and collecting thalli after induction expression;

(3) Crushing the thalli obtained in the step (2), and centrifuging to collect supernatant;

(4) And (4) purifying the supernatant obtained in the step (3) to obtain the recombinant human collagen.

7. The method for preparing recombinant human collagen according to claim 6, wherein in step (1), said recipient cells comprise E.coli expression strains;

preferably, in step (2), the method for inducing treatment comprises inducing with IPTG;

preferably, the IPTG induction adopts IPTG with the final concentration of 0.5-1.5 mM;

preferably, the OD value at the time of the induction treatment is 0.6 to 0.8;

preferably, the temperature of the induction treatment is 36-38 ℃, the rotating speed of the induction treatment is 120-320 rpm, and the time of the induction treatment is 4-6 h;

preferably, in the step (3), the cell bodies are disrupted by a method comprising the steps of: resuspending the thalli by a PB solution, carrying out high-pressure homogenization, centrifugally collecting supernatant fluid and filtering;

preferably, the mass ratio of the bacteria to the PB solution is 1 (5-50);

preferably, the molar concentration of the PB solution is 10-30 mM;

preferably, the high-pressure homogenization conditions are as follows: breaking the thalli for 2-4 times under the condition of 600-1000 bar;

preferably, the rotation speed of the centrifugation is 12000-16000 rpm, and the time of the centrifugation is 40-60 min;

preferably, the aperture of the filter membrane used for filtration is 0.2-0.8 μm;

preferably, in step (4), the purification method comprises: performing adsorption purification by using a nickel ion column, eluting by using a washing buffer solution and an elution buffer solution in sequence, and then performing fine purification on the eluate by using a phenyl column;

preferably, the impurity washing buffer solution is a 10-30 mM PB solution;

preferably, the elution buffer is 10-30 mM Tris solution;

preferably, the elution buffer used for fine purification is a 10-30 mM Tris solution.

8. Use of the recombinant human collagen of claim 1 or 2 for polypeptide display.

9. Use of the recombinant human collagen of claim 1 or 2 for the preparation of a medicament or cosmetic for the treatment of skin lesions.

10. A composition for treating skin injury and repairing skin, wherein the composition for treating skin injury and repairing skin comprises the recombinant human collagen of claim 1 or 2;

preferably, the composition for treating skin injury and repairing skin comprises the following components in parts by weight: 0.05 to 0.5 portion of recombinant human collagen, 0.05 to 0.5 portion of blue coptin, 2 to 5 portions of bletilla gum, 0.5 to 1.5 portions of fibroin and 90 to 100 portions of water.

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