Recombinant human type XXI collagen and its preparation method and application

Abstract

The present invention provides a recombinant human type XXI collagen protein and a preparation method and application thereof, and relates to the field of genetic engineering technology. The present invention obtains a recombinant human type XXI collagen protein sequence through protein sequence analysis and a large number of experimental tests. The collagen protein recombinantly expressed by this sequence can detect the absorption of characteristic peaks of circular dichroism in vitro, proving that it has a triple helix structure. At the same time, the present invention also uses a cell culture of human fibroblasts to evaluate the recombinant human type XXI collagen protein molecule with the triple helix structure, and determines its good promoting effect on the synthesis of human body's own collagen and elastin.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a recombinant human type XXI collagen protein and a preparation method and application thereof. Background Art

[0002] As an important natural biological protein, collagen can be widely used in many fields such as chemical industry, medicine, food, cosmetics, etc. It is especially suitable for the preparation of various biological devices. It is the most ideal source of biological materials and has broad application prospects. The collagen sold on the market is mainly collagen extracts obtained by treating animal tissues with acid, alkali and enzymatic methods. During the processing of this type of extract, the natural structure of collagen is basically destroyed, and it is severely degraded, resulting in the loss of its biological activity; the extracted collagen peptides are of different lengths, uneven properties, unstable quality and the risk of viral infection; at the same time, the amino acid sequences of animal and human collagen are quite different, which can lead to immune rejection and allergic symptoms.

[0003] Type XXI collagen belongs to the fibril-associated collagen with interrupted triple helix (FACIT) family of collagens. Type XXI collagen can copolymerize with fibrillar collagen through its C-terminal collagen domain and mediate protein-protein interactions through its N-terminal non-collagenous domain, thereby acting as a molecular bridge in the extracellular matrix. Type XXI collagen is expressed in the heart, placenta, stomach, jejunum, skeletal muscle, kidney, lung, pancreas and lymph nodes.

[0004] Due to the constraints of legal ethics, human type XXI collagen has only been studied at the scientific research level. As a result, there are no commercialized type XXI collagen products from humans or other animals on the market, which further limits the research and application of human type XXI collagen. Therefore, there are currently no manufacturers producing human-like type XXI collagen on the market.

[0005] At present, the main way to produce collagen is through biotechnology such as genetic engineering, through fermentation and purification to finally obtain the corresponding product. This method is more efficient than natural extraction, and can also obtain a larger yield, further achieving animal free. However, compared with natural extraction, collagen produced by biotechnology such as genetic engineering cannot obtain a triple helix structure. Generally speaking, the triple helix structure is more in line with the form of human collagen and can better exert its efficacy.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] One of the purposes of the present invention is to provide a recombinant human type XXI collagen, related biomaterials, preparation methods and applications. The recombinant human type XXI collagen has a triple helix structure and is more suitable for human application, so as to at least solve one of the technical problems existing in the prior art.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The present invention provides a recombinant human type XXI collagen, wherein the recombinant human type XXI collagen has an amino acid sequence as described in any one of (i) to (iii) below:

[0010] (i) the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1;

[0011] (ii) the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2;

[0012] (iii) the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 3.

[0013] The present invention provides a tandem protein of recombinant human type XXI collagen, wherein the amino acid sequence of the tandem protein is formed by repeated tandem connection of n core units, wherein n is a natural number greater than 1, and the core unit has an amino acid sequence as described in any one of (i) to (iii) below:

[0014] (i) the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1;

[0015] (ii) the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2;

[0016] (iii) the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3;

[0017] Preferably, n is selected from any value between 2 and 5.

[0018] The present invention provides an isolated nucleic acid encoding the above-mentioned recombinant human type XXI collagen or a tandem protein of recombinant human type XXI collagen;

[0019] Preferably, the nucleic acid has a nucleotide sequence as described in any one of (I) to (III) below:

[0020] (I) the nucleotide sequence set forth in SEQ ID NO:4, or a nucleotide sequence having at least 85% sequence identity to SEQ ID NO:4;

[0021] (II) the nucleotide sequence set forth in SEQ ID NO:5, or a nucleotide sequence having at least 85% sequence identity to SEQ ID NO:5;

[0022] (III) the nucleotide sequence shown in SEQ ID NO:6, or a nucleotide sequence having at least 85% sequence identity with SEQ ID NO:6.

[0023] The present invention provides a recombinant vector carrying the above nucleic acid.

[0024] Furthermore, the vector is an Escherichia coli system expression vector;

[0025] Preferably, the E. coli system expression vector includes pET.

[0026] The present invention provides a recombinant host cell, which carries the above-mentioned nucleic acid or contains the above-mentioned recombinant vector;

[0027] Preferably, the recombinant host cell is selected from eukaryotic cells or prokaryotic cells, preferably Escherichia coli.

[0028] The present invention provides a method for preparing the above-mentioned recombinant human type XXI collagen, comprising the following steps:

[0029] (a) fermenting and culturing the engineered bacteria;

[0030] (b) extracting and purifying the fermentation supernatant to obtain the recombinant human type XXI collagen.

[0031] The present invention provides a product, which comprises at least one of the above-mentioned recombinant human type XXI collagen, a tandem protein of recombinant human type XXI collagen, or the recombinant human type XXI collagen prepared by the above-mentioned preparation method;

[0032] Preferably, the product comprises a lyophilized powder.

[0033] The present invention provides the use of the above-mentioned recombinant human type XXI collagen, the tandem protein of recombinant human type XXI collagen or the recombinant human type XXI collagen prepared by the above-mentioned preparation method in any field of chemical industry, medicine, food and cosmetics.

[0034] The present invention also provides the use of the above-mentioned recombinant human type XXI collagen, the tandem protein of recombinant human type XXI collagen, or the recombinant human type XXI collagen prepared by the above-mentioned preparation method in the preparation of cosmetics having any of the following functions:

[0035] (A) Reduce skin damage;

[0036] (B) Increase or promote the increase of elastin content in the skin.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention obtains three recombinant human type XXI collagen sequences through protein sequence analysis and a large number of experimental tests. The collagen recombinantly expressed with these sequences can detect the absorption of characteristic peaks of circular dichroism in vitro, proving that it has a triple helix structure. Moreover, after any combination of the above different recombinant human type XXI collagen sequences, they also have a triple helix structure.

[0039] 2. Compared with long-chain proteins, small molecules made from human type 21 collagen peptides can more easily enter the dermis, which is beneficial for skin absorption and enhances its efficacy.

[0040] 3. At the same time, the present invention also uses human fibroblast cell culture to evaluate the above-mentioned recombinant human type XXI collagen molecule with a triple helical structure, and determines its good promoting effect on the synthesis of human body's own collagen and elastin. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 This is a circular dichroism test graph of the sample collagen powder provided in Example 1 of the present invention;

[0043] Figure 2 This is a graph showing the results of the MTT assay provided in Example 1 of the present invention;

[0044] Figure 3 This is a graph showing the results of type I collagen determination provided in Example 1 of the present invention;

[0045] Figure 4 This is a circular dichroism test graph of the sample collagen powder provided in Example 2 of the present invention;

[0046] Figure 5 This is a graph showing the results of the MTT assay provided in Example 2 of the present invention;

[0047] Figure 6 This is a graph showing the results of type I collagen determination provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0048] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0049] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The structure of natural collagen can be a triple helix, in which three polypeptide chains together form a spiral coil. A single polypeptide chain consists of a repeating triplet amino acid sequence designated as Gly-XY. X and Y can be any amino acids, and the first amino acid is glycine. The concentrations of the amino acids proline and hydroxyproline are high in collagen. The most common triplet is glycine-proline-hydroxyproline (Gly-Pro-Hyp), which accounts for about 10.5% of the triplets in collagen. The recombinant collagen currently on the market is a polypeptide sequence and a triple helix structure cannot be obtained. Therefore, the ability to reduce skin damage, increase, promote, and stimulate the increase of elastin in the skin is inferior to animal-extracted collagen.

[0052] Type 21 collagen (AAI43866) is 957aa in length. In terms of its amino acid composition, the first 445 amino acid sequences are protein anchoring and binding regions. Positions 447 to 933 are glycine-rich regions. In theory, the triple helix structure of type 21 collagen is mainly formed by this part. Through sequence analysis and a large number of tests, the inventors have summarized the following key sites, whose functional regions are mainly composed of the following three parts: (i) to (iii) any one of the amino acid sequences:

[0053] (i) the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1;

[0054] (ii) the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2;

[0055] (iii) the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 3.

[0056] It can be understood that these three parts are independent helical regions; and in the three-dimensional structure, there are corner regions with other structures, which are independent of each other. Therefore, the recombinant human type XXI collagen with a triple helical structure protected by the present invention can be a single amino acid sequence shown in SEQ ID NO:1, a single amino acid sequence shown in SEQ ID NO:2, a single amino acid sequence shown in SEQ ID NO:3, or a combination of the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:2, a combination of the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:3, a combination of the amino acid sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, or a common combination of the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3. In addition, the order of combination is not limited, and any combination order can achieve the technical effect of the present invention.

[0057] The function of a protein requires an orderly arrangement of its amino acid residues. The active function of the recombinant human type XXI collagen in the present invention is based on at least one specific amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and a certain proportion of homology (greater than 85%) may still achieve the same or similar biological activity.

[0058] It should be noted that the term "identity" or similar terms in the present invention refers to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. The percentage is purely statistical, and the differences between the two sequences may (but not necessarily) be randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually performed after optimal alignment, relative to a fragment or "comparison window" to identify the local region of the corresponding sequence. "Identity" includes amino acid sequences that have at least 85% (for example, but not limited to 85%, 86%, 87%, 88%, 89%, 90%, 92%, 95% or higher) identity with the amino acid sequence shown in SEQ ID NO.1 of the present invention.

[0059] It is understandable that the above-mentioned sequence with more than 85% identity can be an amino acid sequence that is modified to a certain extent by amino acid insertion, substitution, addition, deletion, etc. on the basis of at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO:2, and SEQ ID NO:3, and still has the same active function.

[0060] The amino acid insertion refers to inserting an amino acid residue at an appropriate position of at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO:2, and SEQ ID NO:3. The inserted amino acid residues may be adjacent to each other in whole or in part, or none of the inserted amino acids may be adjacent to each other.

[0061] The amino acid substitution refers to that one or several amino acid residues (including several consecutive amino acid residues or several non-continuous amino acid residues) at a certain position of at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO:2, and SEQ ID NO:3 are replaced by other amino acid residues.

[0062] The amino acid addition refers to adding amino acids at the C-terminus or N-terminus of at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO:2, and SEQ ID NO:3.

[0063] The amino acid deletion refers to the deletion of 1, 2, 3 or more amino acids from at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO:2, and SEQ ID NO:3.

[0064] Based on the beneficial effects of the recombinant human type XXI collagen provided by the present invention, the present invention also provides a tandem protein of the recombinant human type XXI collagen, wherein the amino acid sequence of the tandem protein is formed by repeated tandem connection of n core units, wherein n is a natural number greater than 1, and the core unit has an amino acid sequence as described in any one of (i) to (iii) below:

[0065] (i) the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1; (ii) the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2; (iii) the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 3. The tandem protein of the recombinant human type XXI collagen has the same or similar activity function as the recombinant human type XXI collagen.

[0066] In an optional embodiment, n is selected from any value between 2 and 5, for example, n is 2, 3, 4 or 5.

[0067] According to another aspect of the present invention, an isolated nucleic acid is also provided, which encodes the above-mentioned recombinant human type XXI collagen or a tandem protein of recombinant human type XXI collagen.

[0068] As used herein, "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acids include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The nucleic acid encodes the above-mentioned recombinant human type XXI collagen, optionally encoding a sense strand or an antisense strand. Nucleic acids can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0069] In an optional embodiment, the nucleic acid has a nucleotide sequence as described in any one of (I) to (III) below:

[0070] (I) the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence having at least 85% (for example, but not limited to 85%, 86%, 87%, 88%, 89%, 90%, 92%, 95% or more) sequence identity to SEQ ID NO:4;

[0071] (II) the nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 85% (for example, but not limited to 85%, 86%, 87%, 88%, 89%, 90%, 92%, 95% or more) sequence identity to SEQ ID NO:5;

[0072] (III) the nucleotide sequence shown in SEQ ID NO:6, or a nucleotide sequence having at least 85% (for example, but not limited to 85%, 86%, 87%, 88%, 89%, 90%, 92%, 95% or more) sequence identity with SEQ ID NO:6.

[0073] It can be understood that the nucleotides shown in SEQ ID NO:4 encode the protein of the amino acid sequence shown in SEQ ID NO:1, the nucleotides shown in SEQ ID NO:5 encode the protein of the amino acid sequence shown in SEQ ID NO:2, and the nucleotides shown in SEQ ID NO:6 encode the protein of the amino acid sequence shown in SEQ ID NO:3.

[0074] According to another aspect of the present invention, a recombinant vector is also provided, which carries the above-mentioned nucleic acid.

[0075] The vector is well known to those skilled in the art, including but not limited to plasmid, phagemid, cosmid, artificial chromosome, etc. In an optional embodiment, the vector contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0076] In an optional embodiment, the vector is an E. coli system expression vector, and typically the E. coli system expression vector includes pET.

[0077] According to another aspect of the present invention, a recombinant host cell is also provided, which carries the above-mentioned nucleic acid or contains the above-mentioned recombinant vector.

[0078] When the recombinant host cell carries the nucleic acid provided by the present invention, the nucleic acid can be integrated into a vector, such as a plasmid, and replicated and / or expressed independently of the cell chromosome genome; the nucleic acid can also be integrated into the cell chromosome genome.

[0079] When the recombinant host cell contains the recombinant vector provided by the present invention, the recombinant vector is replicated and / or expressed in the recombinant host cell.

[0080] The recombinant host cell in the present invention may refer to a single cell, a cell line, or a culture containing cells. The recombinant host cell in the present invention includes its offspring, which may not be completely identical to the primary cell due to natural, accidental or intentional mutations, and may differ from the primary cell in morphology and / or in genomic DNA. The cell may be a natural cell or a transformant. The present invention does not limit the specific types of the above-mentioned recombinant host cells provided, including but not limited to eukaryotic or prokaryotic cells, and more specifically including but not limited to bacterial, fungal, plant, insect or animal cells.

[0081] In some specific embodiments, the recombinant host cell is Escherichia coli.

[0082] According to another aspect of the present invention, there is also provided a method for preparing the above-mentioned recombinant human type XXI collagen, comprising the following steps:

[0083] (a) fermenting and culturing the engineered bacteria;

[0084] (b) extracting and purifying the fermentation supernatant to obtain the recombinant human type XXI collagen.

[0085] The preparation method has simple process, convenient operation and is suitable for large-scale production.

[0086] According to another aspect of the present invention, there is also provided a product, which comprises at least one of the above-mentioned recombinant human type XXI collagen, a tandem protein of recombinant human type XXI collagen, or the recombinant human type XXI collagen prepared by the above-mentioned preparation method;

[0087] Preferably, the product comprises a lyophilized powder.

[0088] According to another aspect of the present invention, there is also provided the use of the above-mentioned recombinant human type XXI collagen, the tandem protein of recombinant human type XXI collagen or the recombinant human type XXI collagen prepared by the above-mentioned preparation method in any field of chemical industry, medicine, food and cosmetics, for example, it can be, but not limited to, drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics or health products, etc.

[0089] In addition, according to the key verification test of the present invention, the present invention also provides the use of the above-mentioned recombinant human type XXI collagen, the tandem protein of recombinant human type XXI collagen, or the recombinant human type XXI collagen prepared by the above-mentioned preparation method in the preparation of cosmetics having any of the following functions:

[0090] (A) Reduce skin damage;

[0091] (B) Increase or promote the increase of elastin content in the skin.

[0092] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0093] Example 1

[0094] 1. Strain construction and fermentation

[0095] Take E. coli BL21 for example. The nucleotide sequence shown in SEQ ID NO.5 is constructed into the multiple cloning site of the expression vector pET. When constructing the strain, necessary start codons and stop codons are added before and after the gene, and the T7 promoter is used for expression. The amino acid sequence expressed is shown in SEQ ID NO.2.

[0096] The fermentation method is carried out according to the standard fermentation method of E. coli BL21 (DE3).

[0097] The fermentation culture of genetically engineered Escherichia coli and the induction and expression of recombinant human collagen are as follows:

[0098] (1) Pick a single colony of genetically engineered Escherichia coli with a larger colony morphology from the LB plate, place it in 10 ml of LB medium, and culture it at 37°C and 220 rpm for 12-16 hours;

[0099] (2) Inoculate the bacterial solution into 2×YT medium at a ratio of 1:100 and culture at 37°C for about 3 hours. When the OD600 is between 0.4 and 0.6, add IPTG at a final concentration of 0.5 mM for induction. Continue to culture at 16°C for 20 hours. Collect the cells by centrifugation and store at -20°C or proceed to the next purification step immediately.

[0100] 2. Protein Extraction and Purification

[0101] The extraction method follows conventional protein extraction operations, and the specific steps are as follows:

[0102] 1. Experimental operation

[0103] (1) Resuspend (1 L) the bacterial pellet in about 50 ml of phosphate buffer (pH 8) (40 mM sodium dihydrogen phosphate, 500 mM sodium hydroxide), centrifuge at 8000 rpm, 4°C for 10 min, and collect the pellet; wash the cells repeatedly for a total of 2 times; then lyse the cells using a high-pressure lysis instrument and centrifuge at 13000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0104] (2) When the cell lysis solution is at about 20°C, 35% (m / m) sulfuric acid is added dropwise while stirring rapidly to adjust the pH value of the material to between 3.0 and 3.1. During the addition, large pieces of white matter can be seen to precipitate, which is a normal phenomenon. After the addition of sulfuric acid, the large pieces of white matter disappear, and the material changes from a slightly yellow turbid liquid to a milky white turbid liquid. After the pH adjustment is completed, continue to stir slowly for 30 minutes, centrifuge the liquid at 8000 rpm and 4°C for 10 minutes, collect the supernatant, and discard the precipitate. The supernatant should be a light yellow clear and transparent liquid. Take a 5 ml sample of the centrifuged supernatant, dilute it 5 times to 25 ml, adjust the pH value to 7.0, and pour the remaining supernatant back. Use a gun tip to pick up an appropriate amount of the precipitate and perform electrophoresis detection together with the supernatant. If there are still flocs floating on the surface of the supernatant after centrifugation, consider filtering or increasing the centrifugation time to remove them to avoid clogging the membrane package.

[0105] (3) The supernatant after acid precipitation is diluted by adding 4 ml of purified water to 1 ml of supernatant, and then adjusted to pH 3.5 with 2M sodium hydroxide solution (the pH is controlled between 3.5-3.6). The membrane system is operated, and a 30 nm PVDF material is used for microfiltration. Under the conditions of motor frequency of 40 Hz, filtration pressure of 0.4 MPa, and filtration temperature of about 25°C, the volume of the liquid to be treated is first compressed to 2 L (the minimum volume required for membrane system filtration), and then 2 L of purified water is added to the circulation tank, and then compressed to 2 L. This is repeated 5 times, and the permeate is collected. 30 ml of the retentate is sampled and adjusted to pH 7.0 and then electrophoresed, and the retentate is discarded; the permeate should be a light yellow clear and transparent liquid; if the next step is not temporarily performed, the liquid can be temporarily stored in a 4°C refrigerator;

[0106] (4) Run the membrane system, filter the microfiltration permeate with a PES ultrafiltration membrane of 30,000 molecular weight cutoff at a motor frequency of 40 Hz, a filtration pressure of 0.6 MPa, and a filtration temperature of about 25°C. First, compress the liquid to 2 L (the minimum volume required for membrane system filtration), add 2 L of purified water to the circulation tank, and then compress it to 2 L; repeat this process 5 times, top wash the retentate to remove salt and pigment, collect the retentate, sample 30 ml of the permeate and retentate respectively, adjust the pH to 7.0, and perform electrophoresis (after the retentate is sampled and electrophoresed, pour it back), and temporarily store the permeate at 4°C until the electrophoresis results are confirmed and discarded;

[0107] (5) The ultrafiltration retentate has an electrophoretic purity of more than 95%, and is frozen at -80°C for more than 12 hours and then freeze-dried.

[0108] 2. Detection of protein molecules

[0109] The obtained type 21 collagen was tested for molecular weight and purity using SDS PAGE. The specific process is: take 40μl of the purified protein solution, add 10μl 5× protein loading buffer (250mM TrisHCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in 100℃ boiling water and boil for 10min, then add 10μl to each well in SDS PAGE protein gel, run it at 160V for 1h, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. Finally, compare the molecular weight to confirm the correctness of the protein.

[0110] 3. Triple helix structure determination

[0111] The purified protein was sent to Huace Testing Agency for protein sequence and circular dichroism spectrum analysis according to Huace internal standard procedures (results as shown in Figure 1 ) for determination.

[0112] 3. Protein effect verification

[0113] The cell culture of human fibroblasts was used to evaluate the above-mentioned truncated human type 21 collagen molecules to determine their promoting effect on the body's own collagen and elastin synthesis. The cell culture of human fibroblasts was also used to determine the viability of human fibroblasts after exposure to truncated type 21 collagen.

[0114] A 2% w / w stock solution of cut collagen was prepared from the cut collagen described above. Aliquots from the 2% stock solution of cut collagen were then used in the following experiments.

[0115] 1. Preparation of Fibroblasts

[0116] Fibroblasts were seeded into 0.5 ml of fibroblast growth medium (FGM) in each well of a 24-well plate and incubated overnight at 37 ± 2 ° C and 5 ± 1% CO2. On the second day, the culture medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5 ml of fresh FGM. The cells were grown until confluent, and the culture medium was replaced every 48 to 72 hours. After confluence was reached, the cells were treated with DMEM supplemented with 1.5% FBS for 24 hours to eliminate any effects of growth factors contained in the normal culture medium. After a 24-hour clearance period, cells were treated with type 21 collagen extracted and dissolved in FGM containing 1.5% FBS at a specified concentration. Transforming growth factor β (TGFβ) (20 ng / ml) was used as a positive control for collagen and elastin synthesis. Untreated cells (negative control) received only DMEM containing 1.5% FBS. The cells were incubated for 48 hours, and the cell culture medium was collected at the end of the incubation period and frozen (75 ° C) or measured immediately. Materials were tested in triplicate.

[0117] 2. MTT assay

[0118] The MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay is a colorimetric assay used to determine the metabolic activity of cells. Changes in cell number are assessed by the MTT assay. When cells are exposed to MTT, reduction of MTT by mitochondria in living cells results in the formation of insoluble purple formazin crystals, which are then extracted from the cells with isopropanol and quantified spectrophotometrically. Non-living cells are unable to reduce MTT and therefore fail to produce purple formazin crystals. The intensity of the purple color is directly proportional to the number of living cells (metabolically active cells). The intensity of the purple color is directly proportional to the metabolic activity of the cells and inversely proportional to the toxicity of the test material.

[0119] After the 2-day incubation discussed above, the cell culture medium (see above) was removed and the fibroblasts were washed twice with PBS to remove any residual jellyfish glycogen molecules. After the last wash, 500 μl of DMEM supplemented with 0.5 mg / ml MTT was added to each well and the cells were incubated for 1 hour at 37 ± 2 ° C and 5 ± 1% CO2. After incubation, the DMEM / MTT solution was removed and the cells were washed once again with PBS, and then 0.5 ml of isopropanol was added to the wells to extract the purple formazan crystals. 200 μl of isopropanol extract was transferred to a 96-well plate and the plate was read at 540 nm using isopropanol as a blank. The average MTT absorbance value of the negative control cells was calculated and used to represent 100% cell viability. Then, the individual MTT absorbance values ​​of the cells undergoing various treatments were divided by the average value of the negative control cells and expressed as a percentage to determine the changes in cell viability caused by each treatment.

[0120] To determine the optimal concentration of collagen to use, perform an experiment by using a specified aliquot of a 2% cut collagen stock solution in the assay. For example, in a sample testing a "10% collagen solution," use an aliquot of 2% cut collagen in an amount sufficient to provide 10% of the assay volume. For a total assay volume of 1.0 ml, use 100 μl of 2% cut collagen stock solution. “10% collagen solution” is 0.2% collagen, “5% collagen solution” is 0.1% collagen, “1% collagen solution” is 0.02% collagen, “0.5% collagen solution” is 0.01% collagen, “0.1% collagen solution” is 0.002% collagen, “0.05% collagen solution” is 0.001% collagen, “0.01% collagen solution” is 0.0002% collagen, and “0.005% collagen solution” is 0.0001% collagen.

[0121] Extracted human type 21 collagen showed protective effects by increasing cell viability in human fibroblasts. Figure 2 As can be seen, the highest values ​​were observed in the MTT assay when fibroblasts were exposed to 0.02% to 0.2% cut human type 21 collagen.

[0122] 3. Procollagen synthesis

[0123] Fibroblasts are the main source of extracellular matrix peptides, including structural proteins, collagen and elastin. Procollagen is a large peptide synthesized by fibroblasts in the dermis of the skin and is a precursor of collagen. When the peptide is processed to form mature collagen, the propeptide portion is cut off (type I C-peptide). Then, both mature collagen and type I C-peptide fragments are released into the extracellular environment. When collagen is synthesized, type I C-peptide fragments accumulate in the tissue culture medium. Since the stoichiometric ratio between the two parts of the procollagen peptide is 1:1, the determination of type I C-peptide will reflect the amount of synthesized collagen. Type I C-peptide can be determined by an ELISA-based method.

[0124] A series of type I C-peptide standards ranging from 0 ng / ml to 640 ng / ml were prepared. Next, ELISA microplates were prepared by removing any unwanted strips from the plate frame and then adding 100 μl of peroxidase-labeled anti-procollagen type I C-peptide original antibody to each well used for the assay. 20 μl of sample (collected tissue culture medium) or standard was then added to the appropriate wells, the microplate was covered, and it was incubated at 37°C for 3 ± 0.25 hours. After incubation, the wells were aspirated and washed three times with 400 μl of wash buffer. After removing the last wash, 100 μl of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as a chromogen) was added to each well and the plate was incubated at room temperature for 15 ± 5 minutes. After incubation, 100 μl of stop solution (1N sulfuric acid) was added to each well and the plate was read at 450 nm using a microplate reader.

[0125] To quantify the amount of each substance present, a standard curve was generated using the known concentrations of each substance. Regression analysis was performed to establish a line that best fits these data points. The absorbance values ​​for the test material and the untreated sample were used to estimate the amount of each substance present in each sample. The results are shown in Table 1. Figure 3 As shown, within the concentration range of 0.02%-0.2%, collagen can effectively promote the release of type I C-peptide fragments.

[0126] Example 2

[0127] 1. Strain Construction

[0128] This embodiment creates two collagen combinations, namely, a combination of SEQ ID NO:1 fragment and SEQ ID NO:2 fragment (Col21-12), and a combination of SEQ ID NO:1 and SEQ ID NO:3 (Col21-13) fragments. When constructing the corresponding expression strain, the start codon ATG is added before each sequence, and the gene synthesis company (GenScript) synthesizes and constructs it into the MCS region of the expression vector pET. The T7 promoter is used for expression.

[0129] Among them, the nucleotide sequence of Col21-12 is shown in SEQ ID NO:7, and the nucleotide sequence of Col21-13 is shown in SEQ ID NO:8.

[0130] The constructed plasmids were named pET-Col21-12 and pET-Col21-13. The two plasmids were electroporated into commercial Escherichia coli BL21 (DE3) to obtain Col21-12 and Col21-13 strains.

[0131] 2. Protein extraction, purification and detection

[0132] 1. Extraction and purification operation

[0133] (See Example 1 for details)

[0134] 2. Detection of protein molecules

[0135] The obtained type 21 collagen was tested for molecular weight and purity using SDS PAGE. The specific process is: take 40μl of the purified protein solution, add 10μl 5× protein loading buffer (250mM TrisHCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in 100℃ boiling water and boil for 10min, then add 10μl to each well in SDS PAGE protein gel, run it at 160V for 1h, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R 250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) for decolorization. Finally, compare the molecular weight to confirm the correctness of the protein.

[0136] 3. Triple helix structure determination

[0137] The purified protein was sent to Huace Testing Agency for protein sequence and circular dichroism spectrum analysis according to Huace internal standard procedures (results as shown in Figure 4 ) for determination.

[0138] 3. Protein effect verification

[0139] The cell culture of human fibroblasts was used to evaluate the above-mentioned intercepted human type 21 collagen molecules to determine their promoting effect on the body's own collagen and elastin synthesis. The cell culture of human fibroblasts was also used to determine the viability of human fibroblasts after exposure to collagen.

[0140] A 2% w / w collagen stock solution was prepared from the above collagen. Aliquots from the 2% collagen stock solution were then used in the following experiments.

[0141] 1. Preparation of Fibroblasts

[0142] Fibroblasts were seeded into 0.5 ml of fibroblast growth medium (FGM) in each well of a 24-well plate and incubated overnight at 37 ± 2 ° C and 5 ± 1% CO2. On the second day, the culture medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5 ml of fresh FGM. The cells were grown until confluent, and the culture medium was replaced every 48 to 72 hours. After confluence was reached, the cells were treated with DMEM supplemented with 1.5% FBS for 24 hours to eliminate any effects of growth factors contained in the normal culture medium. After a 24-hour clearance period, cells were treated with type 21 collagen dissolved in FGM containing 1.5% FBS at a specified concentration. Transforming growth factor β (TGF-β) (20 ng / ml) was used as a positive control for collagen and elastin synthesis. Untreated cells (negative control) received only DMEM containing 1.5% FBS. The cells were incubated for 48 hours, and the cell culture medium was collected and frozen (-75 ° C) or measured immediately at the end of the incubation period. Materials were tested in triplicate.

[0143] 2. MTT assay

[0144] After the 2-day incubation discussed above, the cell culture medium (see above) was removed and the fibroblasts were washed twice with PBS to remove any residual jellyfish glycogen molecules. After the last wash, 500 μl of DMEM supplemented with 0.5 mg / ml MTT was added to each well and the cells were incubated for 1 hour at 37 ± 2 ° C and 5 ± 1% CO2. After incubation, the DMEM / MTT solution was removed and the cells were washed once again with PBS, and then 0.5 ml of isopropanol was added to the wells to extract the purple formazan crystals. 200 μl of isopropanol extract was transferred to a 96-well plate and the plate was read at 540 nm using isopropanol as a blank. The average MTT absorbance value of the negative control cells was calculated and used to represent 100% cell viability. Then, the individual MTT absorbance values ​​of the cells undergoing various treatments were divided by the average value of the negative control cells and expressed as a percentage to determine the changes in cell viability caused by each treatment.

[0145] In this embodiment, the experiment is performed by using a specified aliquot of a 2% collagen stock solution in the assay. For example, in a sample testing a "10% collagen solution", an aliquot of 2% collagen sufficient to provide 10% of the assay volume is used. For a total assay volume of 1.0 ml, 100 μl of 2% collagen stock solution is used. That is, "10% collagen solution" is 0.2% collagen, "5% collagen solution" is 0.1% collagen, "1% collagen solution" is 0.02% collagen, "0.5% collagen solution" is 0.01% collagen, "0.1% collagen solution" is 0.002% collagen, "0.05% collagen solution" is 0.001% collagen, and "0.01% collagen solution" is 0.0002% collagen.

[0146] Human collagen type 21 showed protective effects by increasing cell viability in human fibroblasts. Figure 5 As can be seen in Figure 2, the highest values ​​were observed in the MTT assay when fibroblasts were exposed to 0.02% to 0.2% human type 21 collagen.

[0147] 3. Procollagen synthesis

[0148] Fibroblasts are the main source of extracellular matrix peptides, including structural proteins, collagen and elastin. Procollagen is a large peptide synthesized by fibroblasts in the dermis of the skin and is a precursor of collagen. When the peptide is processed to form mature collagen, the propeptide portion is cut off (type I C-peptide). Then, both mature collagen and type I C-peptide fragments are released into the extracellular environment. When collagen is synthesized, type I C-peptide fragments accumulate in the tissue culture medium. Since the stoichiometric ratio between the two parts of the procollagen peptide is 1:1, the determination of type I C-peptide will reflect the amount of synthesized collagen. Type I C-peptide can be determined by an ELISA-based method.

[0149] A series of type I C-peptide standards ranging from 0 ng / ml to 640 ng / ml were prepared. Next, ELISA microplates were prepared by removing any unwanted strips from the plate frame and then adding 100 μl of peroxidase-labeled anti-procollagen type I C-peptide original antibody to each well used for the assay. 20 μl of sample (collected tissue culture medium) or standard was then added to the appropriate wells, the microplate was covered, and it was incubated at 37°C for 3 ± 0.25 hours. After incubation, the wells were aspirated and washed three times with 400 μl of wash buffer. After removing the last wash, 100 μl of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as a chromogen) was added to each well and the plate was incubated at room temperature for 15 ± 5 minutes. After incubation, 100 μl of stop solution (1N sulfuric acid) was added to each well and the plate was read at 450 nm using a microplate reader.

[0150] To quantify the amount of each substance present, a standard curve was generated using the known concentrations of each substance. Regression analysis was performed to establish a line that best fits these data points. The absorbance values ​​for the test material and the untreated sample were used to estimate the amount of each substance present in each sample. The results are shown in Table 1. Figure 6 As shown in the figure, it can be seen that when a collagen solution higher than 5% is used, type I C-peptide has a significant improvement compared to the negative control, indicating that type 21 collagen (Col21-12 and Col21-13) in the present invention can effectively promote procollagen synthesis. In actual use, it is highly likely that human collagen synthesis can be effectively improved.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Recombinant human type XXI collagen, characterized in that: The amino acid sequence of the recombinant human type XXI collagen is shown in SEQ ID NO:

2.

2. An isolated nucleic acid, characterized in that Encoding the recombinant human type XXI collagen according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

5.

4. A recombinant vector, characterized in that: Carrying the nucleic acid according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The vector is an E. coli system expression vector.

6. The recombinant vector according to claim 5, characterized in that The E. coli system expression vector includes pET.

7. A recombinant host cell, characterized in that Carrying the nucleic acid according to claim 2 or 3 or comprising the recombinant vector according to any one of claims 4 to 6.

8. The recombinant host cell according to claim 7, characterized in that The recombinant host cell is selected from eukaryotic cells or prokaryotic cells.

9. The recombinant host cell according to claim 8, characterized in that The recombinant host cell is Escherichia coli.

10. The method for preparing recombinant human type XXI collagen according to claim 1, characterized in that: The steps include: (a) fermenting and culturing the engineered bacteria; (b) extracting and purifying the fermentation supernatant to obtain the recombinant human type XXI collagen.

11. A product, characterized in that The product comprises at least one of the recombinant human type XXI collagen described in claim 1 or the recombinant human type XXI collagen prepared by the preparation method described in claim 10.

12. The product according to claim 11, characterized in that The product includes a lyophilized powder.

13. Use of the recombinant human type XXI collagen according to claim 1 or the recombinant human type XXI collagen prepared by the preparation method according to claim 10 in the preparation of any one of chemical products, medicines, foods and cosmetics.

14. Use of the recombinant human type XXI collagen according to claim 1 or the recombinant human type XXI collagen prepared by the preparation method according to claim 10 in preparing cosmetics having the function of increasing or promoting the increase of the content of elastin in the skin.

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