Recombinant I-type collagen as well as preparation method and application thereof

Abstract

The invention discloses recombinant I-type collagen as well as a preparation method and application thereof, and relates to the field of biology. The amino acid sequence of the recombinant I type collagen comprises A-Bn-A; wherein the amino acid sequence of A has at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO: 2, the amino acid sequence of B has at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO: 3, and n is an integer and is greater than or equal to 1. A sequence in the recombinant I-type collagen is derived from natural collagen, immunological rejection is avoided when the recombinant I-type collagen is used for a human body, and the recombinant I-type collagen can be correctly folded into a triple helix in vitro and has good stability and biological activity; the method has the advantage of being better in effect when being applied to preparation of products such as medicines, medical instruments, biological materials, tissue engineering products, cosmetics and skin care products.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a recombinant type I collagen and a preparation method and application thereof. Background Art

[0002] Collagen is an extracellular protein, a fibrous protein made of three peptide chains twisted into a spiral shape. Collagen is the most abundant protein in the human body, accounting for 25% to 30% of the total body protein. Collagen is rich in amino acids such as glycine, proline, and hydroxyproline that the human body needs. It is mainly found in human skin, bones, eyes, teeth, tendons, and internal organs (including the heart, stomach, intestines, and blood vessels). Its function is to maintain the shape and structure of the skin and tissues and organs, and it is also an important raw material for repairing damaged tissues.

[0003] The main source of collagen is still animal collagen, such as seafood such as fish and byproducts of livestock processing such as cattle and pigs, which is obtained through acid, alkali, enzyme and other extraction processes. Animal collagen prepared using this traditional method has certain viral risks, and is prone to allogeneic rejection when applied to the human body, limiting the application of collagen.

[0004] With the development of biotechnology, microorganisms are used as chassis cells to achieve the expression and production of collagen. The structure of collagen is relatively complex. In organisms, the synthesis and modification of collagen starts from procollagen, undergoes many chemical changes such as hydroxylation, glycosylation, and mutual cross-linking, and is subject to complex regulation by a variety of biological enzymes to form a triple helix structure. There are several problems in the existing technology in the structural design of recombinant collagen: first, the hydroxylation function of proline and lysine is missing; second, many designed recombinant collagens are characterized after expression and found to not have a triple helix structure, and are very unstable and easily form precipitation, which restricts the further application of recombinant collagen.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant type I collagen and a preparation method and application thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a recombinant type I collagen, wherein the amino acid sequence of the recombinant type I collagen comprises: AB n-A; wherein the amino acid sequence of A has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of B has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and n is an integer and ≥1.

[0009] In a second aspect, an embodiment of the present invention provides a recombinant type I collagen, which contains the recombinant type I collagen described in the above embodiment, and wherein at least one proline is replaced by hydroxyproline.

[0010] In a third aspect, an embodiment of the present invention provides an isolated nucleic acid molecule encoding the recombinant type I collagen described in the aforementioned embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides a recombinant vector or a host cell containing the recombinant vector, wherein the recombinant vector includes the nucleic acid molecule described in the above embodiment.

[0012] In a fifth aspect, an embodiment of the present invention provides a method for preparing the recombinant type I collagen as described in the aforementioned embodiment, which comprises: culturing the host cell described in the aforementioned embodiment.

[0013] In a sixth aspect, an embodiment of the present invention provides a composition, the active ingredient of which includes the recombinant type I collagen described in the above embodiment.

[0014] In a seventh aspect, an embodiment of the present invention provides a product, the active ingredient of which includes the recombinant type I collagen described in the above embodiment.

[0015] In an eighth aspect, embodiments of the present invention provide the use of the recombinant type I collagen as described in the preceding embodiments in promoting cell adhesion or preparing a product having the effect of promoting cell adhesion.

[0016] In a ninth aspect, embodiments of the present invention provide use of the recombinant type I collagen as described in the preceding embodiments in the preparation of a product that promotes skin wound repair and / or tissue regeneration.

[0017] The present invention has the following beneficial effects:

[0018] The embodiment of the present invention provides a recombinant type I collagen, the amino acid sequence of which comprises: AB n-A; wherein the amino acid sequence of A has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of B has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3, and n is an integer and ≥ 1. The sequence in the recombinant type I collagen is derived from natural collagen, and there will be no immune rejection reaction when used in the human body. The recombinant type I collagen can be correctly folded into a triple helix in vitro, has good stability and biological activity, and has the advantage of better effect when applied to the preparation of products such as drugs, medical devices, biomaterials, tissue engineering products, cosmetics and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The purity test results of recombinant type Ⅰ humanized collagen C1-2G; 1: C1-2, 2: C1-2 (L593);

[0021] Figure 2 is the hydroxylation rate of C1-2 (L593) relative to human type I collagen (sigma-C7774);

[0022] Figure 3 This is the secondary map result of a fragment in recombinant type Ⅰ humanized collagen C1-2G;

[0023] Figure 4 This is the circular dichroism identification result of recombinant type Ⅰ humanized collagen C1-2G;

[0024] Figure 5 The circular dichroism identification result of C1-2 (L593);

[0025] Figure 6 The thermal stability of recombinant type Ⅰ humanized collagen C1-2G;

[0026] Figure 7 The thermal stability of C1-2 (L593);

[0027] Figure 8 The effect of samples on 3T3 cell adhesion;

[0028] Fig. 9 The figure shows the statistical results of 3T3 cell adhesion. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] In one aspect, an embodiment of the present invention provides a recombinant type I collagen, wherein the amino acid sequence of the recombinant type I collagen comprises: AB n -A; wherein the amino acid sequence of A has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of B has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and n is an integer and ≥1.

[0031] In AB n -In the structural formula shown in A, B is a repeating unit, and the sequence of n repeating units is directly connected; 2 A's are respectively connected to the N-terminus and C-terminus of the n repeating unit sequence, and the sequence is a terminal sequence peptide segment that helps collagen fold into a triple helix.

[0032] On the one hand, A and B in the recombinant type I collagen provided by the embodiment of the present invention are derived from human type I collagen (amino acid sequence as shown in SEQ ID NO: 1), which avoids the inherent difficulties of animal-derived collagen, significantly improves the biocompatibility of recombinant type I collagen, and solves the problem of immunogenicity; on the other hand, the recombinant type I collagen strictly complies with the Gly XY repeating sequence 100%, and there is no risk of disease caused by mutation; further, the structural design of the recombinant collagen is more purposeful, can be correctly folded into a triple helix in vitro, has better stability and biological activity, and can be widely used in skin care products, skin repair dressings, medical beauty and health foods and other fields.

[0033] In some embodiments, n can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a range between any two thereof. When n≥2, each repeating unit (B) is directly connected.

[0034] In some embodiments, n is 6-10.

[0035] "Percent sequence identity" herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after the candidate sequence is aligned and, if necessary, spaces are introduced to obtain maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for determining percent amino acid sequence identity can be accomplished in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0036] In some embodiments, the at least 80% sequence identity can specifically be any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, or a range between any two of them.

[0037] In some embodiments, the A may have one or more amino acid substitutions, deletions, insertions and / or additions based on the sequence shown in SEQ ID NO: 2. Alternatively, the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID No. 2, 1, 2 or 3 amino acids are replaced by amino acids with similar or similar properties to form a peptide. These conservative amino acid substitutions can be performed as shown in Table 1.

[0038] Table 1 Conservative substitution of amino acids

[0039]

[0040]

[0041] In some embodiments, B may have one or more amino acid substitutions, deletions, insertions and / or additions based on the sequence shown in SEQ ID NO: 3. Alternatively, the substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID No. 3, 1, 2 or 3 amino acids are replaced by amino acids with similar or similar properties to form a peptide. These conservative amino acid substitutions can be performed as shown in Table 1.

[0042] In some embodiments, the amino acid sequence of the recombinant type I collagen includes: an amino acid sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO:4 and having the same or similar cell adhesion effect as the sequence shown in SEQ ID NO:4. For example, one or more amino acids are substituted, deleted, inserted and / or added based on the sequence shown in SEQ ID NO:4, and the multiple amino acids can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. Optionally, the substitution can be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID No.4, 1, 2 or 3 amino acids are replaced by amino acids with similar or similar properties. These conservative amino acid substitutions can be performed with reference to those shown in Table 1.

[0043] In some embodiments, the at least 80% sequence identity can specifically be any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, or a range between any two of them.

[0044] In some embodiments, the amino acid sequence of the recombinant type I collagen includes: the sequence shown in SEQ ID NO:4.

[0045] In some embodiments, the amino acid sequence of the recombinant type I collagen further includes: a tag for purification or labeling.

[0046] In some embodiments, the amino acid sequence of the recombinant type I collagen is as shown in SEQ ID NO:5.

[0047] All amino acids in the polypeptide sequence of this article may be L-type amino acids, and one or more (such as 2-5, 2-4 or 2-3) of the amino acids may also be replaced with amino acids with D-type conformation, artificially modified amino acids, rare amino acids existing in nature, etc., to improve the bioavailability, stability and / or antiviral activity of the polypeptide. Among them, D-type amino acids refer to amino acids corresponding to L-type amino acids that constitute proteins; artificially modified amino acids refer to common L-type amino acids that constitute proteins that have been modified by methylation, phosphorylation, etc.; rare amino acids existing in nature include uncommon amino acids that constitute proteins and amino acids that do not constitute proteins, such as hydroxyproline, 5-hydroxylysine, methylhistidine, γ-aminobutyric acid, homoserine, etc.

[0048] In another aspect, a recombinant type I collagen comprises the recombinant type I collagen described in any of the above embodiments, wherein at least one proline is replaced by hydroxyproline.

[0049] In some embodiments, the recombinant type I collagen contains the recombinant type I collagen described in any of the aforementioned embodiments, wherein 10% to 20% of proline is replaced by hydroxyproline. Specifically, the amount of proline replaced by hydroxyproline is any one of 10%, 12%, 14%, 16%, 18%, 20%, or any two of the range.

[0050] In another aspect, an embodiment of the present invention provides an isolated nucleic acid molecule encoding the recombinant type I collagen described in any of the aforementioned embodiments.

[0051] In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:6.

[0052] On the other hand, an embodiment of the present invention provides a recombinant vector, which includes the nucleic acid molecule described in any of the above embodiments.

[0053] In an embodiment of the present invention, the recombinant vector is known in the field of vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements. The recombinant vector includes a sequence suitable for introduction into a cell. For example, the vector can be an expression vector, in which the coding sequence of the protein is controlled by its own cis-acting regulatory elements, and the design of the vector is convenient for gene integration or gene replacement of the host cell. It should be understood by those of ordinary skill in the art that in the present invention, "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleotide sequences. Suitable phage and viral vectors include, but are not limited to: λ-phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus and lentivirus, etc.

[0054] In some embodiments, the recombinant expression vector comprises: any one of a pET series vector, a shuttle vector, a phage and a viral vector.

[0055] In some embodiments, the pET series vectors include pET-28a.

[0056] On the other hand, an embodiment of the present invention provides a host cell, which includes the recombinant vector described in any of the above embodiments.

[0057] In some embodiments, the host cell can be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell, such as fungi or yeast.

[0058] Based on the amino acid sequence of recombinant type I collagen disclosed in the present invention, it is easy for those skilled in the art to conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the recombinant type I collagen. For example, the recombinant type I collagen can be separated and purified from the culture products of recombinant cells that can recombinantly express the recombinant type I collagen as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibodies or functional fragments thereof of the present invention, they all fall within the scope of protection of the present invention.

[0059] On the other hand, an embodiment of the present invention provides a method for preparing the recombinant type I collagen as described in any of the aforementioned embodiments, comprising: culturing the host cell described in any of the aforementioned embodiments to induce protein expression.

[0060] In some embodiments, the culturing is performed in a shaker at 220 rpm and 37°C.

[0061] In some embodiments, the specific procedure for inducing protein expression may be: cooling to 16° C. and adding IPTG; optionally, the concentration of the IPTG is 1 mM.

[0062] In some embodiments, after inducing protein expression, the preparation method further comprises: collecting the expressed recombinant type I collagen; and purifying the recombinant type I collagen.

[0063] Optionally, the specific procedure for collecting the expressed recombinant type I collagen may be: centrifuging the product obtained by inducing protein expression at 4000 rpm for 10-20 min at 4°C, resuspending the precipitate in a solution containing 1×PBS, adding an inhibitor and then ultrasonically crushing it, and then centrifuging it at 15000-18000 rpm for 20-40 min at 4°C. The purification method may be purification using a Ni affinity chromatography column.

[0064] Optionally, the method for preparing recombinant type I collagen may include: (1) constructing genetically engineered Escherichia coli; (2) fermenting and culturing the genetically engineered Escherichia coli; (3) inducing and expressing the protein; and (4) purifying the protein.

[0065] Optionally, in the above step (1), the construction of the Escherichia coli genetically engineered bacteria can be carried out by the following steps: the expression vector containing the nucleotide sequence of the recombinant type I collagen described in any of the above embodiments is transferred into the Escherichia coli competent cells BL21 (DE3), and positive Escherichia coli genetically engineered bacteria are screened.

[0066] Optionally, in the above steps (2) and (3), the fermentation culture of Escherichia coli genetically engineered bacteria and the induction and expression of proteins can be carried out by the following steps: a. Pick a single colony of the preferred Escherichia coli genetically engineered bacteria, place it in an LB liquid culture medium (10 ml) containing kanamycin, culture it at 37°C, 220 rpm overnight, and then transfer it to an LB liquid culture medium (1 L) containing kanamycin for culture until the OD value is between 0.6 and 0.8, then cool it to 16°C; b. Add IPTG at a final concentration of 1 M for induction, and collect the bacteria after culturing for 12-16 hours, that is, collect the bacteria by centrifugation at 4000 rpm for 20 min at 4°C.

[0067] In the above step (4), the protein purification can be carried out by the following steps: a. resuspending the bacteria with a buffer solution (1×PBS, pH 7.4), and then disrupting the bacteria. After disruption, centrifuging at 16000 rpm for 30 minutes at 4°C, and collecting the supernatant; b. using a Ni affinity column to bind the protein, and then washing the Ni column with a buffer solution, then rinsing the impurities with a washing buffer solution containing 20 mM imidazole, and then eluting the target protein with an elution buffer solution containing 300 mM imidazole; c. desalting the target protein.

[0068] The embodiment of the present invention provides an E. coli expression system for the recombinant human type I collagen described in any of the aforementioned embodiments, which has high expression level, high purity and is easy to purify.

[0069] It should be understood that those skilled in the art can replace the above-mentioned E. coli strains with other expression strains as host cells.

[0070] On the other hand, an embodiment of the present invention provides a composition, the active ingredient of which includes the recombinant type I collagen described in any of the above embodiments.

[0071] On the other hand, an embodiment of the present invention provides a product, the active ingredient of which includes the recombinant type I collagen described in any of the above embodiments.

[0072] In some embodiments, the article is a material that provides cells with space for adhesion, support and / or growth and migration, or a material that serves as a channel for transporting nutrients and / or metabolic products;

[0073] In some embodiments, the product includes any one of a drug, a medical device, a biomaterial, a tissue engineering product, a cosmetic, and a skin care product;

[0074] In some embodiments, the medicament comprises a skin repair dressing.

[0075] On the other hand, an embodiment of the present invention provides a use of the recombinant type I collagen as described in any of the above embodiments in promoting cell adhesion or preparing a product having the effect of promoting cell adhesion.

[0076] In addition, an embodiment of the present invention provides the use of the recombinant type I collagen as described in any of the aforementioned embodiments in the preparation of a product that promotes skin wound repair and / or tissue regeneration.

[0077] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0078] Example 1 Preparation of recombinant humanized type I collagen C1-2G and C1-2 (L593)

[0079] 1. Construction of C1-2G and C1-2(L593) gene expression vectors

[0080] 1.1 The full-length protein sequence of the recombinant type I humanized collagen C1-2G used in this example is the amino acid sequence shown in SEQ ID No.4, with a total length of 198aa. In order to purify the C1-2G protein after its expression, this example connected MHHHHHHHHGSGSGSENLYFQ to the N-terminus of the amino acid sequence shown in SEQ ID No:4 to obtain the following amino acid sequence: MHHHHHHHHGSGSGSENLYFQGPPGPPGPPGPPGPPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGDAGAPGAPGSQGAPGLQGMPGPPGPPGPPGPPGPP (SEQ ID No: 5), with a total length of 219aa. The coding sequence corresponding to the amino acid sequence was codon-optimized for the codons of Escherichia coli, and the corresponding gene had a full length of 660 bp (SEQ ID No. 6).

[0081] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the gene fragments, and the synthesized gene fragments were inserted into the pET 28a expression vector through the HindⅢ and BamHI restriction sites to obtain the corresponding recombinant expression plasmid.

[0082] 1.2 Hydroxylase L593 (amino acid sequence is the same as SEQ ID NO.6 in patent CN202310276337.4, nucleic acid sequence is the same as SEQ ID NO.7 in patent CN202310276337.4) was used to construct plasmid pETduet-1-C1-2G-L593 expressing collagen C1-2G and proline hydroxylase L593.

[0083] 2. Construction of genetically engineered Escherichia coli:

[0084] Construction of engineered bacteria expressing recombinant type I humanized collagen C1-2G: The successfully constructed expression plasmid was transferred into Escherichia coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the competent E. coli cells BL21 (DE3) from the -80°C refrigerator and place them on ice. When they are half melted, take out 1 μL of the expression plasmid expressing C1-2G and add it to the competent E. coli cells BL21 (DE3) in a clean bench; (2) Place the mixture on ice for 5 minutes, then heat shock it in a 42°C water bath for 60 seconds, take it out and place it on ice for 5 minutes; (3) Add 500 μL of liquid LB culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / ml kanamycin) in a clean bench and resuscitate at 37°C and 220 rpm for 60 minutes; (4) Take 200 μL of the resuscitated bacterial solution and evenly spread it on an LB plate containing kanamycin; (5) Place the plate in a 37°C incubator for overnight culture until colonies of uniform size grow.

[0085] Construction of an engineered bacterium expressing recombinant type I humanized collagen C1-2 (L593) with proline hydroxylation: The constructed plasmid expressing C1-2G and proline hydroxylase L593 was transferred into the competent Escherichia coli cell BL21 (DE3). The specific process can refer to the specific process of the engineered bacterium expressing recombinant type I humanized collagen C1-2G.

[0086] 3. Induced expression of recombinant humanized type Ⅰ collagen C1-2G and C1-2 (L593):

[0087] The specific process is: pick the colonies on the plate to 10ml LB liquid culture medium (add 50μg / ml kanamycin when expressing C1-2G, add 100μg / ml ampicillin when expressing C1-2(L593)), put it in a 37℃ shaker, culture at 220rpm for about 12h, transfer it to 1L culture medium, culture at 37℃, 220rpm until OD600 is 0.6-0.8, add 500μL IPTG (final concentration 0.5mM), and induce at 16℃, 160rpm for 16-20h.

[0088] 4. Protein purification:

[0089] The specific process is as follows: the bacterial solution is divided into centrifuge bottles, centrifuged at 4000 rpm and 4°C for 20 minutes, and the bacterial cells are collected. The collected bacterial cells are resuspended in binding buffer (1×PBS pH7.4), and then ultrasonically disrupted. The bacterial solution after cell disruption is divided into centrifuge bottles, centrifuged at 16000 rpm and 4°C for 30 minutes, and the supernatant is collected.

[0090] Add the supernatant to a 1ml Ni affinity resin gravity column. After the flow-through is complete, add 8 column volumes of binding buffer to wash the Ni column. Then add 8 column volumes of washing buffer (1×PBS pH7.4, 20mM imidazole) to wash the Ni column. Finally, use 8 column volumes of elution buffer (1×PBS pH7.4, 300mM imidazole) to elute the target protein. Dialyze overnight at 4°C to obtain the desalted target protein.

[0091] Example 2 Biological evaluation of recombinant humanized type I collagen C1-2G and C1-2 (L593)

[0092] 1. Purity test of recombinant humanized type Ⅰ collagen C1-2G and C1-2 (L593)

[0093] The purity of the obtained C1-2G and C1-2 (L593) proteins was detected by SDS PAGE. The specific process was as follows: 20 μL of the purified protein solution was taken, 5 μL of 5× protein loading buffer (250 mM Tris HCl pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added, and the solution was boiled in 100°C boiling water for 5 min, and then 10 μL of the solution was added to the SDS PAGE protein gel per well, and after electrophoresis at a voltage of 250 V for 0.5 h, the protein was stained with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R 250, 25% ethanol, 10% glacial acetic acid) for 3 min, and then decolorized with a protein decolorizing solution (10% acetic acid, 5% ethanol).

[0094] The results are as follows Figure 1 shown.

[0095] 2. Proline hydroxylation detection of C1-2 (L593)

[0096] The absorbance at OD560nm was detected using the HYP detection kit (Solarbio, BC0250), including the positive control (standard HYP), human type I collagen (sigma, C7774) and sample C1-2 (L593). The samples were processed according to the instructions of the HYP detection kit, and the feasibility of the experiment was confirmed based on the results of the blank control and positive control. The relative hydroxylation rate of proline in protein C1-2 (L593) was calculated with the hydroxyproline content of human type I collagen as 100%. The results are shown in Figure 2 The hydroxyproline content in protein C1-2 (L593) is about 30%-40% of the hydroxyproline content in human type I collagen, that is, the relative hydroxylation rate of proline in protein C1-2 (L593) reaches about 30%-40%. It should be noted that the hydroxylation rate of proline in protein C1-2 (L593) is about 10% to 20%.

[0097] 2. Mass spectrometry identification results of recombinant humanized type Ⅰ collagen C1-2G

[0098] The theoretical size of the recombinant type I humanized collagen C1-2G provided in the embodiment of the present invention is 19.2 kD. The SDS PAGE gel results show that the size of the target protein is about 55 kD. The sample is identified by mass spectrometry after gel cutting and in-gel enzymatic digestion. The specific operation is as follows:

[0099] Cut the target band of about 1×1mm from the gel block, put it into an EP tube, and wash it three times with water; decolorize the gel block with a decolorizing solution (ACN / 50mM ammonium bicarbonate 1:1 mixture) at a constant temperature of 37°C until the color is completely removed; add 100% ACN to cover the gel particles, dehydrate until the gel block turns white and hard, absorb the ACN, and vacuum dry for 5 minutes until the tube is completely dry; add α-chymotrypsin working solution (790μl ACN:200mM ammonium bicarbonate:H2O=1:2:7+10μl 2000μg / ml), place it at 4°C for 20min, adjust the liquid level to just cover the swollen gel particles after the gel particles are fully swollen, invert the EP tube, and incubate at 37°C for 12-16 hours; add an appropriate amount (5-10μL) of 10% FA to terminate the reaction; aspirate the reaction solution, store it in an EP tube, and add 50% ACN / 0.1% FA was passed through the particles, and the mixture was incubated at 37℃ for 45min, vortexed, centrifuged, and the extract was aspirated and added to the reaction solution EP tube, and repeated once; the EP tube with the reaction solution and the two extracts was centrifuged and concentrated to dryness, 25μl of 0.1% FA sample was dissolved, and centrifuged at 12000rpm for 20min, and 20μl of the supernatant was taken and loaded into the sample machine. The results were analyzed. The analysis results showed that the sample was C1-2G. Figure 3 Shown is the secondary map result of one of the fragments.

[0100] 3. Circular dichroism identification results of recombinant humanized type Ⅰ collagen C1-2G and C1-2(L593)

[0101] Recombinant humanized type I collagen C1-2G and C1-2 (L593) were diluted to 0.2 mg / ml with PBS and the absorption at 200-260 nm was scanned using Chirascan plus (Applied Photophysics). Figure 4 , Figure 5 As shown, both C1-2G and C1-2 (L593) have the circular dichroism spectrum of the standard collagen triple helix, that is, there is a negative peak before 200nm and a positive peak near 220nm. The circular dichroism results show that the MilliDegree value at 221nm is 3.96511, and the MilliDegree value of C1-2 (L593) at 224nm is 0.247316.

[0102] 4. Thermal stability of recombinant humanized type Ⅰ collagen C1-2G and C1-2(L593)

[0103] Recombinant type I humanized collagen C1-2G and C1-2 (L593) were diluted to 0.5 mg / ml with PBS, respectively. After being treated at different temperatures for 1 hour, they were centrifuged at 12000 rpm for 20 minutes. 20 μL of the sample in the supernatant was taken, 5 μL of 5× protein loading buffer was added, and the mixture was boiled in 100°C boiling water for 5 minutes. Then 10 μL of each well was added to SDS PAGE protein gel. After electrophoresis at 250 V for 0.5 hours, Coomassie Brilliant Blue staining solution was used, and then protein decolorization solution was used for decolorization. The results of C1-2G are as follows: Figure 6 As shown, 1, 2, 3, 4, 5, 6, 7, and 8 represent C1-2G that has not been heated, and 1-2G samples that have been heated at 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 1 hour, respectively, which indicates that C1-2G has good solubility and excellent high temperature resistance.

[0104] C1-2(L593) results are as follows Figure 7 As shown, 1, 2, 3, 4, 5, 6, 7, and 8 represent C1-2 (L593) without heat treatment and C1-2 (L593) samples after heating at 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 1 hour, respectively, which shows that C1-2 (L593) has good solubility and excellent high temperature resistance.

[0105] 5. Bioactivity Determination of Recombinant Humanized Type I Collagen C1-2G and C1-2 (L593)

[0106] Determine the concentration of the protein solution. Dilute the sample 50 times, use NanoDrop to measure the UV absorption of the sample at 215nm and 225nm, and calculate the protein concentration using the empirical formula C (μg / mL) = 144×(A215 A225)×50. Note that the test must be performed when A215 < 1.5. After the protein concentration is tested, use buffer (1×PBS pH7.4) to adjust the concentration of all proteins to be tested to 0.05mg / mL.

[0107] Set up blank wells, PBS wells, control wells (i.e., human type I collagen), and collagen sample wells. Add 100 μL of PBS, human type I collagen, and C1-2G collagen solution to the 96-well plate and let stand at room temperature for 60 minutes. Add 100 μL of 1×10 6 / ml of 3T3 cells in good culture condition were cultured in a 37°C cell culture incubator with 95% air, 5% CO2 and sterile water for 60 minutes. The supernatant was gently turned upside down and washed three times with PBS. The remaining liquid in the well plate was sucked out as much as possible with a pipette tip in the last wash. The OD was detected using the LDH detection kit (Roche, 4744926001). 492nm and OD 690nm The absorbance.

[0108] According to the value of the blank control, the absorbance corresponding to each well can be calculated. The calculated absorbance value is proportional to the number of cells attached to the wall. The calculation formula is as follows: Sample absorbance value = test well A 492nm -A 690nm -Blank Well A 492nm -A 690nm The calculated absorbance value can reflect the number of cells attached to the wall, and the number of cells attached to the wall can reflect the activity of collagen. The higher the activity of the protein, the better the external environment can be provided to the cells in a short time, helping the cells to adhere to the wall.

[0109] The sample adhesion results are as follows Figure 8 The statistical results are shown in Fig. 9 As shown in the results, it can be seen that the adhesion activity of the recombinant humanized type I collagen C1-2G and C1-2 (L593) of the present invention is better than that of the commercial humanized type I collagen (sigma-C7774).

[0110] The sequence information involved is as follows.

[0111]

[0112]

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant type I collagen, characterized in that: The amino acid sequence of the recombinant type I collagen includes: AB n -A; wherein the amino acid sequence of A has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of B has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and n is an integer and ≥1.

2. The recombinant type I collagen according to claim 1, characterized in that The amino acid sequence of A is shown in SEQ ID NO: 2; the amino acid sequence of B is shown in SEQ ID NO: 3; Optionally, n is 6 to 10; Optionally, the amino acid sequence of the recombinant type I collagen comprises: an amino acid sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO: 4; Optionally, the amino acid sequence of the recombinant type I collagen includes: the sequence shown in SEQ ID NO: 4; Optionally, the amino acid sequence of the recombinant type I collagen further comprises: a tag for purification or labeling; Optionally, the amino acid sequence of the recombinant type I collagen is as shown in SEQ ID NO:

5.

3. A recombinant type I collagen, characterized in that: It contains the recombinant type I collagen according to claim 1 or 2, wherein at least one proline is replaced by hydroxyproline.

4. An isolated nucleic acid molecule, characterized in that It encodes the recombinant type I collagen according to any one of claims 1 to 3; Optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

6.

5. A recombinant vector or a host cell containing the recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule of claim 4; Optionally, the recombinant vector is a recombinant expression vector; Optionally, the recombinant expression vector includes: any one of pET series vectors, shuttle vectors, phage and virus vectors; Optionally, the pET series vectors include pET-28a.

6. The method for preparing recombinant type I collagen according to any one of claims 1 to 3, characterized in that: It includes: Cultivate the host cell according to claim 5.

7. A composition, characterized in that The active ingredient comprises the recombinant type I collagen according to any one of claims 1 to 3.

8. A product, characterized in that The active ingredient comprises the recombinant type I collagen according to any one of claims 1 to 3; Optionally, the product is a material that provides cells with space for adhesion, support and / or growth and migration, or a material that serves as a channel for transporting nutrients and / or metabolic products; Optionally, the product includes any one of drugs, medical devices, biomaterials, tissue engineering products, cosmetics, and skin care products; Optionally, the medicament comprises a skin repair dressing.

9. Use of the recombinant type I collagen according to any one of claims 1 to 3 in promoting cell adhesion or preparing a product having the effect of promoting cell adhesion.

10. Use of the recombinant type I collagen according to any one of claims 1 to 3 in the preparation of a product for promoting skin wound repair and / or tissue regeneration.

Images