Recombinant III-type humanized collagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to recombinant III-type humanized collagen as well as a preparation method and application thereof. On the basis of the III-type human collagen amino acid sequence, the amino acid sequence is designed by means of bioinformatics and the like, so that the recombinant collagen realizes higher expression efficiency and a triple helix structure on the premise of maintaining high biological activity, a section of brand new collagen sequence containing 180 amino acids is designed, the protein molecular weight is small, and the recombinant collagen has the advantages of high expression efficiency, high yield, high yield and the like. The skin permeability is better. Compared with a commercially available collagen product, the recombinant humanized collagen prepared by the technical scheme has better stability, cell proliferation and migration promotion effect and barrier repair capability, so that the recombinant humanized collagen has good practical application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant type III humanized collagen and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. At present, about 29 different types of collagen have been identified. According to the order of discovery of collagen, it can be divided into type I, type II, type III, etc. Among them, type III collagen is the main collagen in human skin, fascia, and tendons. It is distributed at the junction of the epidermis and dermis, providing elasticity and stress resistance to the skin. In addition, type III collagen is present in high levels in blood vessels. With its good mechanical properties, it maintains the strength and tension of blood vessels and promotes the formation of new blood vessels. It is also effective in promoting wound damage repair and activating coagulation factors to promote coagulation mechanisms. However, with the increase of age, type III collagen continues to accelerate its loss and cannot be regenerated by itself. In recent years, with the gradual deepening of functional activity research, type III collagen has been used as a key raw material in medical cosmetology, medical devices, beauty skin care, food applications and other fields.

[0004] Different collagens can be divided into animal-derived collagen and recombinant collagen according to their sources. Collagen obtained from animal connective tissue has product quality differences due to animal-derived viruses and individual differences in animals, which to a certain extent limits the application and development of new products. Recombinant collagen is a non-natural collagen synthesized by genetic engineering technology. It has higher biological activity and biocompatibility, lower immunogenicity, lower risk of missed pathogens, better water solubility, and can be further processed and optimized. Therefore, its application range is wider and has received strong attention from the market. Most of the recombinant collagen that has been commercialized comes from microbial expression systems; the expression hosts are mainly Escherichia coli and Pichia pastoris. However, the expression level of recombinant collagen from Escherichia coli is generally low, and the various fusion tags contained in the protein sequence may affect the activity of the protein itself, and most of the expressed recombinant collagen cannot form a triple helix structure. Summary of the invention

[0005] In view of the above-mentioned prior art, the inventors have provided a recombinant type III humanized collagen and its preparation method and use after long-term technical and practical exploration. Experiments have proved that the recombinant type III collagen produced by the present invention has high yield and good safety; has better cell proliferation promotion, migration activity and barrier repair biological activity; and has low purification cost, which is suitable for industrial large-scale production. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a recombinant humanized type III collagen, wherein the recombinant humanized type III collagen is any one of the following A1)-A3):

[0008] A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0009] A2) a protein related to the recombinant humanized type III collagen obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1);

[0010] A3) A protein that has more than 85% identity with A1) or A2) and is related to the recombinant humanized type III collagen.

[0011] The recombinant humanized type III collagen has a triple helix structure.

[0012] The second aspect of the present invention provides a polynucleotide capable of encoding the above-mentioned recombinant humanized type III collagen.

[0013] Specifically, the polynucleotide is any one of the following B1) or B2):

[0014] B1) The nucleotide sequence is a DNA molecule described in SEQ ID NO.2;

[0015] B2) A DNA molecule having more than 85% identity with the DNA molecule shown in B1) and having the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or replacing and / or deleting and / or adding one or more nucleotides.

[0016] The third aspect of the present invention provides a vector comprising the above-mentioned polynucleotide.

[0017] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned polynucleotide and the above-mentioned vector; or, the host cell is capable of expressing the above-mentioned recombinant humanized type I and II collagen.

[0018] A fifth aspect of the present invention provides a method for preparing recombinant human type I and II collagen, comprising the steps of:

[0019] The recombinant type III human collagen is expressed by the host cells and then separated and purified to obtain the recombinant type III human collagen.

[0020] The sixth aspect of the present invention provides the use of the above-mentioned recombinant humanized type III collagen, the recombinant humanized type III collagen encoded by the polynucleotide, or the recombinant humanized type III collagen produced by the above-mentioned host cell in the preparation of food, cosmetics or pharmaceutical products.

[0021] A seventh aspect of the present invention provides a cosmetic comprising at least the above-mentioned recombinant humanized type I and II collagen.

[0022] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial technical effects:

[0023] The above technical scheme is based on the amino acid sequence of type III human collagen, and the amino acid sequence is designed by means of bioinformatics and other means, so that the recombinant collagen can achieve higher expression efficiency and triple helix structure under the premise of maintaining high biological activity, and a new collagen sequence containing 180 amino acids is designed, the protein molecular weight is small, and it has better skin permeability. The nucleotide sequence of the recombinant collagen is optimized by the codon preference of Escherichia coli, so as to construct a high-expression recombinant strain. The recombinant collagen produced by the fermentation of the recombinant strain does not have any exogenous amino acids such as labels, and is a recombinant humanized collagen with good safety. The present invention also provides a low-cost and efficient protein purification process, which is suitable for large-scale production. Compared with commercially available collagen products, the recombinant humanized collagen prepared by the above technical scheme has better stability, cell proliferation and migration promoting effect and barrier repair ability, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 The figure is the SDS-PAGE detection diagram of the recombinant bacteria E.coil BL21 / pET28a-F15 after induced expression; wherein, lane M represents the standard protein with a molecular weight of 180 kDa; and lane 1 represents the supernatant of the recombinant bacteria E.coil BL21 / pET28a-F15 cell lysis.

[0026] Figure 2The SDS-PAGE protein electrophoresis diagram of the protein purified after high-density culture of the recombinant bacteria E.coil BL21 / pET28a-F15 in a 20L fermenter; wherein, lane M represents the standard protein with a molecular weight of 180kDa; lane 1 represents the purified F15 collagen.

[0027] Figure 3 It is a bar graph of DPPH free radical scavenging rate of different collagens; wherein, the comparative example represents the comparison experimental group of commercially available recombinant collagen; and F15 represents the experimental group of recombinant humanized collagen prepared by Example 5 of the present invention.

[0028] Figure 4 It is a bar graph of the relative expression of FLG mRNA in HaCaT cells under the action of different collagens; wherein, control group 1 represents the blank control group; control group 2 represents the commercially available recombinant collagen control group; Col represents the recombinant humanized collagen F15 group prepared in Example 5 of the present invention; in the figure, compared with control group 1, P<0.001(***), P<0.0001(****), which is considered to be significantly different.

[0029] Figure 5 This is the circular dichroism spectrum detection diagram of recombinant collagen F15. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] In a typical embodiment of the present invention, a recombinant humanized type III collagen is provided, wherein the recombinant humanized type III collagen is any one of the following A1)-A3):

[0033] A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0034] A2) a protein related to the recombinant humanized type III collagen obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1);

[0035] A3) A protein that has more than 85% identity with A1) or A2) and is related to the recombinant humanized type III collagen.

[0036] The proteins shown in A1)-A3) above can be artificially synthesized, or their encoding genes can be synthesized first and then obtained by biological expression.

[0037] In the present invention, the recombinant humanized type III collagen has a triple helix structure.

[0038] In the present invention, amino acid substitution may refer to the replacement of an amino acid at a certain position in an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide.

[0039] In the present invention, amino acid deletion may refer to deleting 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0040] In the present invention, amino acid addition may refer to adding 1, 2 or 3 or more amino acids at the C-terminus, N-terminus or any position between the C-terminus and the N-terminus of the amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0041] In the present invention, the term "identity" refers to the percentage of identical (i.e., identical) amino acids between two or more polypeptides, which can be calculated by dividing the number of positions containing the same amino acid residue by the total number of amino acid residues in the polypeptide and multiplying by 100.

[0042] Another aspect of the present invention provides a polynucleotide capable of encoding the above-mentioned recombinant humanized type I and II collagen.

[0043] In the present invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function.

[0044] Specifically, the polynucleotide is any one of the following B1) or B2):

[0045] B1) The nucleotide sequence is a DNA molecule described in SEQ ID NO.2;

[0046] B2) A DNA molecule having more than 85% identity with the DNA molecule shown in B1) and having the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or replacing and / or deleting and / or adding one or more nucleotides.

[0047] Another aspect of the present invention provides a vector comprising the above-mentioned polynucleotide.

[0048] In the present invention, "vector" refers to a self-replicating DNA molecule that transfers a DNA fragment (target gene) to a recipient cell in genetic engineering recombinant DNA technology. Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, no free ends; nucleic acid molecules containing DNA, RNA or both; and other polynucleotide species known in the art. One type of vector is a "plasmid" (specifically pET28a), which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of the target gene. Such vectors are referred to herein as "expression vectors." The recombinant expression vector may comprise a form suitable for expressing the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which may be selected based on the host cell for expression and to which the nucleic acid sequence to be expressed may be operably linked.

[0049] Another aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned polynucleotide and the above-mentioned vector; or, the host cell is capable of expressing the above-mentioned recombinant humanized type I and II collagen.

[0050] In the present invention, the term "host cell" refers to a cell into which an expression vector has been introduced. The host cell can be any cell useful in the production of recombinant humanized collagen in the present application. In order to produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli.

[0051] Another aspect of the present invention provides a method for preparing recombinant human type I and II collagen, comprising the steps of:

[0052] The recombinant type III human collagen is expressed by the host cells and then separated and purified to obtain the recombinant type III human collagen.

[0053] The separation and purification steps include impurity protein removal, centrifugation, hollow fiber membrane clarification, ultrafiltration membrane concentration, ion exchange chromatography, etc. By adopting the above separation and purification steps, the purity of the obtained protein is greater than 95%, and the yield is greater than 60%, which is very suitable for industrial production applications.

[0054] Another aspect of the present invention provides the use of the above-mentioned recombinant humanized type III collagen, the recombinant humanized type III collagen encoded by the polynucleotide, or the recombinant humanized type III collagen produced by the above-mentioned host cell in the preparation of food, cosmetics or pharmaceutical products.

[0055] Another aspect of the present invention provides a cosmetic comprising at least the above-mentioned recombinant humanized type I and II collagen. Compared with commercially available collagen products, the recombinant humanized collagen of the present invention has better stability, cell proliferation and migration promoting effect and barrier repair ability, and therefore can be used as a raw material for cosmetics, especially skin care products.

[0056] Of course, the cosmetics may also contain any other raw material ingredients allowed to be added in the cosmetics field, including but not limited to dispersants, skin feel regulators, emulsifiers, emollients, moisturizers, flavor enhancers, colorants, film formers, thickeners and preservatives, etc. Those skilled in the art may choose to add according to actual conditions, and no specific limitation is made here.

[0057] The present invention is further described below in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. The experimental methods used, if not specifically described, are conventional methods. The strains, cells, materials, reagents, etc. used, unless otherwise specified, were obtained from commercial sources.

[0058] Example 1: Recombinant humanized collagen sequence design

[0059] The amino acid sequence of human type III collagen α1 chain protein (GenBank: KAI2526099.1) was obtained from the NCBI database. Through in-depth mining and analysis of the sequence information, and using a variety of technical means such as bioinformatics, the charge characteristics, hydrophilicity and hydrophobicity of amino acids and the three-dimensional spatial structure of proteins were simulated and designed, aiming to ensure that the recombinant collagen maintains high biological activity while achieving higher expression efficiency and optimizing its triple helix structure.

[0060] The recombinant humanized collagen in this embodiment has a total of 180 amino acids, and the specific amino acid sequence is shown in SEQID NO.1. The sequence does not contain any exogenous amino acids such as tags, and includes multiple biologically active sites of human type III collagen. It has high biocompatibility and low risk of use, thereby effectively ensuring the realization of the biological function of the recombinant collagen.

[0061] Example 2: Construction of recombinant E.coil BL21 / pET28a-F15

[0062] Based on the codon preference of E. coli, the coding gene of the recombinant humanized collagen shown in SEQ ID NO.1 in Example 1 was codon optimized, and the optimized coding gene nucleotide sequence is shown in SEQ ID NO.2, which is named F15. The nucleotide sequence of F15 was commissioned to be synthesized by BGI and cloned into the E. coli expression vector pET28a, and then transformed into E.coil TOP10. While ensuring that the reading frame did not shift, the recombinant expression plasmid pET28a-F15 was obtained. The recombinant expression plasmid pET28a-F15 was successfully constructed through DNA sequencing comparison.

[0063] The verified recombinant expression vector pET28a-F15 was transformed into E. coli BL21 (DE3) according to the E. coli operation guide. The recombinant expression transformants were screened using kanamycin antibiotics and further verified by PCR. The recombinant bacteria E.coil BL21 / pET28a-F15 expressing recombinant humanized collagen was successfully constructed.

[0064] Example 3: Induced expression of recombinant E.coil BL21 / pET28a-F15

[0065] A single colony was picked from the recombinant E.coil BL21 / pET28a-F15, placed in LB liquid medium (1% NaCl, 0.5% yeast extract, 1% peptone) containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, and then inoculated into TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K+-1) at a 2% (v / v) inoculum. 2 HPO 4 , 0.22% KH 2 PO 4 , 0.4% glycerol) and continue to culture at 37°C, 200 rpm until OD 600is 0.7. At this time, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.5mM to the culture medium to induce protein expression, and culture at 25°C for 16 hours. After that, collect the bacteria by centrifugation, resuspend the bacteria in Tris buffer (20mM Tris, pH7.5) and perform ultrasonic disruption. The supernatant and precipitate after disruption were respectively subjected to SDS-PAGE detection. According to theoretical prediction, the molecular weight of the recombinant humanized collagen should be 15.6kDa. The results of SDS-PAGE are shown in the figure. Figure 1 As shown, there is an obvious protein expression band near 15.6 kDa. The above results show that the recombinant humanized collagen in this example has been successfully induced to express.

[0066] Example 4: High-density culture of recombinant E.coil BL21 / pET28a-F15 in 5L tank

[0067] The recombinant E.coil BL21 / pET28a-F15 was cultured at high density in a 5L tank. The bacterial solution in the glycerol tube was inoculated into 60mL seed medium (1% peptone, 1% sodium chloride, 0.5% yeast powder) containing 50μg / mL kanamycin at an inoculum of 1‰, and cultured at 37°C and 180rpm for about 15h. The cultured seed liquid was transferred entirely into a 5L fermentation tank containing 3L fermentation medium (3% yeast powder, 1.5% glycerol, 3.25% ammonium sulfate, 0.2% sodium chloride, 1.0% disodium hydrogen phosphate dodecahydrate, 0.31% potassium dihydrogen phosphate, 0.17% magnesium sulfate heptahydrate, 0.02% defoamer, 0.43% trace elements, pH adjusted to 7.0 with sodium hydroxide; 500mL trace element formula: 1.625g boric acid, 0.275g manganese sulfate monohydrate, 0.3325g cupric chloride dihydrate, 0.1375g zinc sulfate monohydrate, 0.211g cobalt chloride, 0.1735g sodium molybdate).

[0068] The initial fermentation parameters were set as follows: stirring speed 150rpm, fermentation temperature 37℃, ventilation 4L / min, dissolved oxygen was adjusted in conjunction with stirring and controlled at 20%, and pH was controlled at 7.0 by automatically adding ammonia water during the fermentation process. When the dissolved oxygen returned to 35%, feeding was started (feed medium formula: 3% ammonium sulfate, 0.22% magnesium sulfate heptahydrate, 30% glycerol, 0.5% trace elements), the initial feeding rate was 0.8mL / min, and the dissolved oxygen was adjusted to 20% by increasing the ventilation volume. When OD 600After the temperature reaches 80, 0.5mM IPTG is added, the temperature is adjusted to 25°C, the dissolved oxygen is controlled at about 30%, and the induction time is 10-20h. After entering the induction time, samples are taken regularly, and the fermentation broth is subjected to SDS-PAGE detection. The grayscale values ​​of the standard protein and the recombinant humanized collagen are determined by BioAnaly biological analysis software. It is calculated that when the recombinant bacteria E.coil BL21 / pET28a-F15 is induced by high-density fermentation in a 5L tank for 15h, the recombinant humanized collagen production is 8.6g / L, achieving a high expression efficiency of the recombinant humanized collagen.

[0069] Example 5: Purification and preparation of recombinant humanized collagen

[0070] The recombinant bacteria E.coil BL21 / pET28a-F15 was fermented in a 5L tank at high density. The fermentation liquid after 15 hours of induction culture was centrifuged at 8000 rpm to obtain bacterial sludge. The bacterial sludge was purified by the following steps to obtain pure recombinant humanized collagen:

[0071] (1) Protein removal: The bacterial solution obtained after resuspending the bacterial slurry to homogenize the bacteria is adjusted to pH 6.5 with low concentration HCl. The bacterial solution is continuously stirred during the acid addition process to denature and precipitate the recombinant humanized collagen;

[0072] (2) Centrifugation: remove the impurities precipitated by acid and adjust the pH of the supernatant to 7.0 with low concentration NaOH;

[0073] (3) Hollow fiber membrane clarification: The centrifugal supernatant obtained in step (2) is filtered through a 250 KDa hollow fiber membrane to remove bacteria and macromolecular impurities;

[0074] (4) Ultrafiltration membrane concentration: Use a 5KDa ultrafiltration membrane to concentrate the feed solution and remove small molecular impurities;

[0075] (5) Ion exchange chromatography: Anion exchange chromatography is used. Recombinant humanized collagen is positively charged. When the sample is loaded on the chromatography column, the flow-through is directly collected. Negatively charged impurities such as impurities such as miscellaneous proteins are adsorbed on the column to achieve the purpose of removing impurities. The obtained flow-through is the purified stock solution. The purity of the protein purified by the above steps is greater than 95%, and the yield is greater than 60%.

[0076] Example 6: Effects of recombinant humanized collagen on cell proliferation and migration

[0077] Effect of recombinant type III collagen F15 on promoting cell proliferation: The results of the CCK-8 method experiment showed that the cell proliferation rates of ESF cells at final concentrations of 0.5 mg / mL and 1.0 mg / mL were 117.3% and 121.8%, which were significantly higher than those of the negative control group (1× maintenance medium), and it is believed that it has a certain effect on promoting ESF cell proliferation.

[0078] The promoting effect of recombinant type III collagen F15 on cell migration: The results of the cell scratch experiment showed that the 24-hour wound healing percentage of the negative control group (NC) was 55.7%, and the 24-hour wound healing percentages (Wound Closure%) of the 0.1 mg / mL and 0.2 mg / mL recombinant type III collagen sample groups were significantly greater than that of the negative control group (NC), approximately 68.8% and 80.5%, respectively. It is believed that the 0.1 mg / mL and 0.2 mg / mL recombinant type III collagen sample groups both have a significant effect in promoting HaCaT wound healing.

[0079] Example 7: Barrier repair experiment

[0080] Human immortalized keratinocytes (HaCaT cells) were collected and cell suspension was prepared with high-glucose DMEM cell culture medium. 2 mL of cell suspension was added to each well of a 6-well plate. The cell number was 2.5 × 10 5 / well. The experiment set up a blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and a recombinant humanized collagen F15 group prepared in Example 5 of the present invention (experimental group), with 3 replicate wells in each group. The 6-well plate was placed in a cell culture incubator (37°C, 5% CO 2 ) for 24 hours. When the cell fusion rate reaches 50% to 60%, the culture medium is discarded. 2 mL of high-glucose DMEM cell culture medium is added to each well of the blank control group; 2 mL of high-glucose DMEM cell culture medium containing the corresponding collagen is added to each well of the other two groups. The concentration of collagen in the high-glucose DMEM cell culture medium is 1 mg / mL. The 6-well plate is placed in an incubator (37°C, 5% CO 2 ) for 24 h, and then each well was washed twice with 2 mL of PBS buffer. According to the Total RNA extraction kit, 1 mL of RNAisoPlus was added, and the cells were lysed by blowing and collecting samples. According to the instructions of the kit, RNA extraction, reverse transcription and fluorescence quantitative PCR detection experiments were carried out to detect the relative expression of mRNA of barrier-related protein filaggrin (FLG). -△△CT Method to calculate.

[0081] The ability of different collagens to promote the mRNA expression of FLG in HaCaT cells Figure 4As shown, compared with the blank control group (control group 1), the commercially available recombinant collagen (control group 2) and the recombinant humanized collagen F15 prepared in Example 5 of the present invention (experimental group) can significantly increase the relative expression of FLG mRNA, among which the recombinant humanized collagen prepared by the present invention has a stronger ability to promote the mRNA expression of FLG than the commercially available recombinant collagen. Therefore, the recombinant humanized collagen of the present invention has obvious skin barrier repair function.

[0082] Example 8: Collagen Circular Dichroism Spectroscopy Experiment

[0083] The obtained recombinant collagen F15 pure product was dissolved in ultrapure water to prepare a solution with a concentration of 0.1 mg / ml. Before spectral analysis, baseline correction was required to remove any system noise or other non-sample related signals. CD detection was performed with a detection wavelength of 180-260 nm.

[0084] The circular dichroism characteristics of collagen triple helix: there is a negative absorption peak near 200nm; there is a weak positive absorption peak near 220nm. The circular dichroism detection diagram of the recombinant collagen F15 in this application is as follows Figure 5 As shown, there is a negative absorption peak at 197nm and a weak positive absorption peak at 218nm, which is consistent with the absorption characteristics of the triple helix.

[0085] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant humanized type III collagen, characterized in that: The recombinant humanized type III collagen is any one of the following A1)-A3): A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; A2) a protein related to the recombinant humanized type III collagen obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1); A3) a protein having more than 85% identity with A1) or A2) and related to the recombinant humanized type III collagen; The recombinant humanized type III collagen has a triple helix structure.

2. A polynucleotide, characterized in that The polynucleotide can encode the recombinant humanized type I and II collagen according to claim 1.

3. The polynucleotide according to claim 2, wherein The polynucleotide is any one of the following B1) or B2): B1) The nucleotide sequence is a DNA molecule described in SEQ ID NO.2; B2) A DNA molecule having more than 85% identity with the DNA molecule shown in B1) and having the same function as the DNA molecule shown in B1) obtained by modifying the nucleotide sequence shown in SEQ ID NO.2 and / or replacing and / or deleting and / or adding one or more nucleotides.

4. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2 or 3.

5. A host cell, characterized in that The host cell contains the polynucleotide according to claim 2 or 3, and the vector according to claim 4; or, the host cell is capable of expressing the recombinant humanized type I and II collagen according to claim 1.

6. A method for preparing recombinant human type I and II collagen, characterized in that: Includes steps: The recombinant human type III collagen is obtained by expressing the host cell described in claim 5 and then isolating and purifying the recombinant human type III collagen.

7. Use of the recombinant humanized type III collagen according to claim 1, the recombinant humanized type III collagen encoded by the polynucleotide according to claim 2 or 3, or the recombinant humanized type III collagen produced by the host cell according to claim 5 in the preparation of food, cosmetics or pharmaceutical products.

8. A cosmetic, characterized in that: The cosmetic contains at least the recombinant humanized type I and II collagen according to claim 1.

9. The cosmetic according to claim 9, characterized in that The cosmetics are skin care products.

10. The cosmetic according to claim 8, characterized in that The cosmetics may also contain any other raw material ingredients permitted to be added in the cosmetics field.

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