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

Abstract

The invention relates to recombinant III-type humanized collagen as well as a preparation method and application thereof, the recombinant III-type humanized collagen comprises an amino acid fragment as shown in SEQ ID No.5, and the recombinant III-type humanized collagen has high cell adhesion property and high thermal stability.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a recombinant type III humanized collagen protein and a preparation method and application thereof. Background Art

[0002] As the core component of the extracellular matrix, collagen has a variety of biological functions, such as maintaining various tissue structures, tissue development and remodeling, affecting cell attachment and proliferation, etc. Among them, type III collagen is the most important and most abundant protein in the human body. Vertebrates have at least 27 types of collagen, with a total of 42 different polypeptides, called α chains. All collagens are composed of three α chains, which are entangled with each other to form a triple helix containing a repeating triplet sequence Gly-XY. Gly is necessary for the formation of hydrogen bonds in collagen. It has no side chains itself, which allows collagen to be tightly packed. At a higher structural level, collagen superhelices will further associate into collagen fibrils. In organisms, the synthesis and modification of collagen begins with tropocollagen, undergoes many chemical changes such as hydroxylation, glycosylation, and mutual cross-linking, and is subject to complex regulation by a variety of biological enzymes. In addition to collagen chains, tropocollagen also contains a globular head and tail. Without these heads and tails, the collagen chains would not fold into a correct triple helix and would lack the biological activity of collagen.

[0003] Denatured and partially degraded collagen has been widely studied in medical applications, from tissue engineering and biomaterials to drug delivery systems, because many medical applications, such as wound healing, cell therapy, bone reconstruction and cosmetic technology, rely on its support and healing qualities. In recent years, the demand for collagen and collagen-derived products in medical cosmetic materials has increased year by year. Animal-derived collagen, which is usually extracted from the skin and bone tissue of cattle, pigs, fish, etc., has been found to have poor biochemical properties, may cause immune rejection and allergic symptoms, and has the risk of infection with viruses such as bovine spongiform encephalopathy (BSE). Therefore, the development of recombinant humanized collagen in animal, plant and microbial recombinant expression systems using transgenic technology is expected to solve many problems in the traditional animal-derived collagen production process. Summary of the invention

[0004] Based on this, it is necessary to provide a recombinant humanized type III collagen with high cell adhesion properties and high thermal stability in one embodiment of the present application.

[0005] The technical solutions include the following:

[0006] Recombinant humanized type III collagen, wherein the recombinant humanized type III collagen comprises the amino acid fragment shown in SEQ ID No.5.

[0007] The nucleic acid molecule comprises the nucleic acid encoding the recombinant humanized type III collagen.

[0008] An expression vector comprising the nucleic acid molecule; optionally, further comprising a nucleic acid encoding the amino acid fragment shown in SEQ ID NO.6.

[0009] In one embodiment, the expression vector comprises a nucleic acid encoding the amino acid fragment shown in SEQ ID NO.7.

[0010] A host cell, which expresses the recombinant humanized type III collagen, contains the nucleic acid molecule or contains the expression vector.

[0011] The method for preparing the recombinant humanized type III collagen comprises culturing the host cells and separating the recombinant humanized type III collagen from the obtained culture product.

[0012] Optionally, the recombinant humanized type III collagen is separated by Ni column and / or anion exchange chromatography.

[0013] In one embodiment, the method further includes a step of enzymatically cleaving the protein; optionally, includes a step of enzymatically cleaving the protein with TEV protease; optionally, does not include an additional step of removing endotoxin; optionally, the purified protein contains less than 0.5EU / mg of endotoxin.

[0014] A product comprising the recombinant humanized type III collagen; optionally, the product comprises a medical device, a tissue engineering product, a cosmetic or a health product; optionally, the recombinant humanized type III collagen is in the form of a lyophilized powder or an aqueous solution; optionally, the product does not contain a component for preventing protein degradation.

[0015] The use of the recombinant type III humanized collagen in the preparation of a product for promoting cell adhesion.

[0016] The application of the recombinant humanized type III collagen in the preparation of tissue repair materials.

[0017] Compared with the traditional technology, this application has the following beneficial effects:

[0018] Compared with traditional recombinant type III collagen, it has higher cell adhesion properties; it has higher stability at 37°C, thus ensuring that the recombinant type III collagen of the present application can maintain structural stability in the human body for a longer period of time and continue to exert its efficacy; in addition, the recombinant type III collagen of the present application is obtained from host cells with higher yield and purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0020] Figure 1 The SDS-PAGE gel results of the expression product;

[0021] Figure 2 The purification of recombinant type III collagen; M is a marker; 1 is the supernatant of cell lysate; 2 is the flow-through after affinity chromatography; 3 is the protein sample after affinity chromatography;

[0022] Figure 3 To compare the cell adhesion activity of recombinant type III collagen;

[0023] Figure 4A-4C The comparison results of cell adhesion activity of recombinant type III collagen composed of different sequence connection orders;

[0024] Figure 5 Figure 1 is the thermal stability of recombinant type III collagen; Figure A shows the degradation of recombinant type III collagen after being placed at 37°C for 1-4 days; Figure B is a comparison of the degradation of two different recombinant collagens of a listed brand (lanes 1 and 2) after being placed at 37°C for 4 days with the recombinant type III collagen in this application example (lane 3).

[0025] Figure 6 Structural prediction for recombinant type III collagen. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0028] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] In this article, the "TEV protease" involved is a common tool enzyme used to remove affinity tags on fusion proteins. It has high specificity and can cut polypeptide chains at specific recognition sequences. Its recognition sequence is usually ENLYFQ↓G / S (↓ indicates the cleavage site). TEV protease has a wide range of applications in biotechnology and biochemistry. For example: in the process of protein purification, it is used to remove fusion tags to obtain pure target proteins, which can minimize the impact on the structure and function of the target protein.

[0030] In this article, "medical devices" refer to instruments, equipment, appliances, in vitro diagnostic reagents and calibrators, materials and other similar or related items used directly or indirectly on the human body.

[0031] In this article, "tissue engineering products" refers to products used for tissue engineering. Tissue engineering is an emerging discipline that combines cell biology and material science to construct tissues or organs in vitro or in vivo.

[0032] One embodiment of the present application provides a recombinant type III humanized collagen (hereinafter referred to as recombinant collagen) comprising n repeats of the sequence shown in SEQ ID No. 1, wherein n is greater than or equal to 1, and optionally is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or 20, wherein when n is an integer greater than or equal to 2, each repeated sequence is directly connected.

[0033] In this application, SEQ ID No.1 is:

[0034] GAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFP.

[0035] Among them, the SEQ ID No.1 repeating sequence is composed of SEQ ID No.2 (GAPGER), SEQ ID No.3 (GERGAPGFRGPAGPNGIPGEKGPAGERGAP) and SEQ ID No.4 (GVMGFP). Among them, SEQ ID No.2 is the integrin binding site domain in type III collagen, which can regulate the adhesion between cells and between cells and extracellular matrix (ECM) through signal transduction. SEQ ID No.4 is the discoidin domain receptor (DDR) domain, which is a receptor tyrosine kinase that can promote cell migration, cell survival, proliferation and differentiation, and remodeling of the extracellular matrix.

[0036] The present application has found that when n has the same value, the cell adhesion and thermal stability performance of the recombinant collagen with n repeats shown in SEQ ID No.1 formed by connecting SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 in sequence is better than the recombinant collagen with n repeats of the sequence formed by connecting SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 in other orders.

[0037] The present application found that the recombinant collagen with n repeats (n=14) of SEQ ID No. 1 is superior to the recombinant collagen with n repeats (n=14, 16) of SEQ ID No. 3 in terms of cell adhesion and thermal stability.

[0038] In a specific example, the recombinant type III humanized collagen contains multiple repeating sequences shown in SEQ ID No. 1, and there is no linker between the repeating sequences. In the application, the linker can be more than one amino acid residue. The application has no limitation on the number of repeating sequences, as long as it can achieve the characteristics verified in the adhesion embodiment.

[0039] In a preferred example, the number of repeating sequences is 14, comprising the recombinant type III humanized collagen shown in SEQ ID No. 5. When n is 14, the recombinant type III humanized collagen has better cell adhesion and better thermal stability at 37°C.

[0040] The recombinant humanized type III collagen of the present application adds an ENLYFQ (SEQ ID No. 6) sequence at its N-terminus during expression, which can be directly cut off by TEV protease to obtain the sequence of SEQ ID No. 5. Preferably, the ENLYFQ (SEQ ID No. 6) sequence is directly connected to the N-terminus, i.e. (SEQ ID NO. 7).

[0041] In a specific example, the recombinant humanized type III collagen comprises an amino acid sequence that is 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID No. 5, and retains the effects of cell adhesion and thermal stability of the amino acid sequence shown in SEQ ID No. 5.

[0042] In a specific example, the recombinant type III humanized collagen comprises a sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID No. 5, as long as the recombinant collagen of the present application retains the cell adhesion and thermal stability effects of the amino acid sequence shown in SEQ ID No. 5. "Multiple" can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

[0043] Amino acid addition refers to adding amino acids to an amino acid sequence, such as the C-terminus or N-terminus of SEQ ID NO.5, as long as the recombinant collagen of the present application retains the cell adhesion and thermal stability effects of the amino acid sequence shown in SEQ ID No.5.

[0044] Amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as SEQ ID NO.5, with another amino acid residue, as long as the recombinant collagen of the present application retains the cell adhesion and thermal stability effects of the amino acid sequence shown in SEQ ID No.5.

[0045] Amino acid insertion refers to inserting an amino acid residue at an appropriate position of an amino acid sequence, such as the sequence of SEQ ID NO. 5. The inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids are adjacent to each other, as long as the recombinant collagen of the present application retains the cell adhesion and thermal stability effects of the amino acid sequence shown in SEQ ID No. 5. In this article, the insertion position of the amino acid is not between each repeating sequence.

[0046] Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, such as the sequence of SEQ ID NO.5, as long as the recombinant collagen of the present application retains the cell adhesion and thermal stability effects of the amino acid sequence shown in SEQ ID No.5.

[0047] In the present application, substitution may be conservative amino acid substitution, which means that compared with the amino acid sequence shown in SEQ ID NO. 5, 3, preferably 2 or 1 amino acids are replaced by amino acids with similar or similar properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

[0048] Table 1

[0049]

[0050] In a specific example, the recombinant humanized type III collagen comprises an amino acid sequence encoded by a nucleotide sequence, the nucleotide sequence hybridizes with the nucleic acid sequence encoding SEQ ID No.5 under stringent conditions, and the amino acid sequence retains the cell adhesion and thermal stability effects of SEQ ID No.5, wherein the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0051] Herein, reference to "moderately stringent conditions", "highly stringent conditions", "highly stringent conditions" or "very highly stringent conditions" describes conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions is provided in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either may be used. For example, specific hybridization conditions are as follows:

[0052] (1) Low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2× SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature can be increased to 55°C);

[0053] (2) moderate stringency hybridization conditions in 6× SSC at about 45°C, followed by one or more washes in 0.2× SSC, 0.1% SDS at 60°C;

[0054] (3) high stringency hybridization conditions at 6× SSC at about 45°C, followed by one or more washes in 0.2× SSC, 0.1% SDS at 65°C;

[0055] (4) Very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2× SSC, 1% SDS at 65°C.

[0056] One embodiment of the present application further provides a nucleic acid molecule, which includes a nucleic acid encoding the above-mentioned recombinant humanized type III collagen. Optionally, the nucleic acid can be DNA or cDNA.

[0057] The nucleic acid molecule may be mainly composed of a nucleic acid sequence encoding the above-mentioned recombinant type III humanized collagen, or may only be composed of a nucleic acid sequence encoding the above-mentioned recombinant type III humanized collagen. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, it will be appreciated by those skilled in the art that nucleic acid molecules of different nucleic acid sequences can encode the same amino acid sequence.

[0058] In a specific example, the nucleic acid sequence encoding the above-mentioned recombinant humanized type III collagen is shown as SEQ ID NO.8.

[0059] One embodiment of the present application also provides an expression vector, comprising the above-mentioned nucleic acid molecule.

[0060] Optionally, the vector also includes a selection of promoters and other regulatory elements, such as enhancer elements. Optionally, the vector also includes sequences suitable for introduction into cells. For example, the vector can be an expression vector, in which the coding sequence of recombinant type III humanized collagen is controlled by its own cis-acting regulatory elements, and the design of the vector facilitates gene integration or gene replacement of host cells, etc.

[0061] Herein, "vector" includes DNA molecules, optionally, plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Optionally, phage and viral vectors include, but are not limited to, lambda phage, EMBL phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine mammary cancer virus, lentivirus, etc. Plasmids include, but are not limited to, PET28a (+).

[0062] In a specific example, the expression vector further comprises a nucleic acid encoding the amino acid fragment shown in SEQ ID NO.6, and the nucleic acid encoding the amino acid fragment shown in SEQ ID NO.6 is directly connected to the 5' end of the nucleic acid encoding the above-mentioned recombinant humanized type III collagen.

[0063] In a specific example, the above expression vector contains a nucleic acid encoding the amino acid fragment shown in SEQ ID NO.7.

[0064] One embodiment of the present application further provides a host cell, which expresses the above-mentioned recombinant humanized type III collagen, contains the above-mentioned polynucleotide, or contains the above-mentioned expression vector.

[0065] In a specific example, the host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae.

[0066] One embodiment of the present application also provides a method for preparing the above-mentioned recombinant type III humanized collagen, comprising culturing the above-mentioned host cells and expressing the protein, and harvesting and purifying the protein. In a specific example, the protein is purified by Ni column and / or anion exchange chromatography. In a specific example, the step of enzymatic protein cleavage is also included. In a specific example, the step of enzymatic protein cleavage with TEV protease is included. In a specific example, no additional step of removing endotoxin is included. In a specific example, the purified protein contains endotoxins below 0.5EU / mg.

[0067] It is understood that recombinant human collagen can also be prepared by conventional methods in the art. For example, the following steps can be used:

[0068] (1) Construction of genetically engineered Escherichia coli;

[0069] (2) Fermentation culture of genetically engineered Escherichia coli;

[0070] (3) Induction and expression of recombinant humanized type III collagen; and

[0071] (4) Purification and optional enzymatic cleavage of recombinant humanized type III collagen.

[0072] In step (1), the construction of the genetically engineered Escherichia coli can be carried out as follows:

[0073] A. Obtain the target gene fragment;

[0074] B. inserting the obtained target gene fragment into the PET-28a(+) expression vector to obtain a recombinant expression plasmid;

[0075] C. The recombinant expression plasmid was transferred into the competent E. coli cells BL21 (DE3), and positive E. coli genetically engineered bacteria were obtained by screening.

[0076] In steps A and B, the fermentation culture of genetically engineered Escherichia coli and the induction and expression of recombinant type III humanized collagen can be performed as follows:

[0077] a) picking a single colony of genetically engineered Escherichia coli from the LB plate, placing it in 10 ml of LB medium and culturing it at 37° C. and 180 rpm for 12-16 hours;

[0078] b) Inoculate the bacterial solution into 2×LB medium at a ratio of 1:100 and culture at 37°C for about 2 hours until OD 600 At 0.4-0.6, IPTG was added at a final concentration of 0.5 mM for induction, and the culture was continued at 37°C for 5 hours. The bacteria were collected by centrifugation.

[0079] In step (4), the purification and enzyme cleavage of recombinant type III humanized collagen can be performed as follows:

[0080] A. Resuspend the bacteria in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0), disrupt by ultrasound, and collect the supernatant by centrifugation;

[0081] B. Recombinant humanized type III collagen was bound to a Ni-NTA affinity column. After the impurities were washed with 10 mM imidazole, TEV protease was added and digested on the column at 4°C for 16 h to obtain the target recombinant humanized type III collagen.

[0082] It is understood that those skilled in the art can replace the above-mentioned E. coli strains with other expression strains as host cells.

[0083] One embodiment of the present application further provides a product, which comprises the above-mentioned recombinant humanized type III collagen. Optionally, the product includes medical equipment, tissue engineering products, cosmetics or health products.

[0084] The recombinant type III humanized collagen of the present application can be used as a Class III medical device for subcutaneous filling, has good structural stability under human body temperature conditions, and can exert a filling effect more continuously.

[0085] In a specific example, the recombinant humanized type III collagen is in the form of a lyophilized powder or an aqueous solution.

[0086] In one specific example, the product does not contain components that prevent protein degradation.

[0087] In a specific example, the product is a product for long-term use, and long-term use means use for more than half a year.

[0088] One embodiment of the present application also provides the use of the above-mentioned recombinant humanized type III collagen in the preparation of a product for promoting cell adhesion.

[0089] One embodiment of the present application also provides the use of the above-mentioned recombinant humanized type III collagen in the preparation of tissue repair materials, wherein the tissue repair materials include materials that promote wound healing.

[0090] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0091] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0092] Example 1 Construction and expression of recombinant humanized type III collagen polypeptide

[0093] 1. The full-length protein sequence of human collagen used is the sequence shown in SEQ ID No.5, with a total length of 588aa and a corresponding gene length of 1764bp. After optimizing the codons of Escherichia coli (nucleotide sequence: SEQ ID NO.8), Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene fragments, and the synthesized polypeptide gene fragments were inserted into the PET28a expression vector through the restriction sites of BamH I (NEB, catalog number: R0136L) and Xho I (NEB, catalog number: R0146L).

[0094] (SEQ ID No.5).

[0095]

[0096] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3). The specific process is:

[0097] (1) Take 1 μL of the plasmid and add it to 100 μL of E. coli competent cells BL21 (DE3) and place it on ice for 30 min.

[0098] (2) The mixture was heat-shocked in a 42°C water bath for 90 seconds and then quickly placed on ice for 2 minutes.

[0099] (3) Add 500 μL of LB without resistance to the mixture and culture at 37°C and 180 rpm for 45 min.

[0100] (4) Take 200 μL of the bacterial solution and spread it evenly on an LB plate containing kanamycin sulfate resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL kanamycin sulfate).

[0101] (5) Incubate the plate upside down in a 37°C incubator for about 16 h until clearly visible colonies appear.

[0102] 2. Pick a single clone from the transformed LB plate and culture it in 10 ml LB (containing 50 μg / mL kanamycin sulfate) medium for 12h-16h, then transfer it to LB medium (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride) at a ratio of 1:100 for expansion culture at 37°C and 180rpm until the bacterial solution OD reaches 600 When the pH was 0.4-0.6, IPTG (Biyuntian, catalog number: ST098-5g) was added at a final concentration of 0.5mM to induce expression. The induction conditions were 37°C and 180rpm for 4h. Finally, the cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next step of purification. After the expression was completed, some cells were detected by SDS-PAGE. The results were as follows Figure 1 As shown in the figure, the expression of recombinant type III collagen. After the recombinant type III collagen is purified, about 1g of pure protein can be obtained from 1L of bacterial liquid.

[0103] 3. Resuspend the bacterial pellet with about 50 ml of Tris-HCl buffer (pH 8.0) (50 mM Tris-HCl, 300 mM NaCl), break the bacteria with an ultrasonic disruptor, and centrifuge at 13,000 rpm for 30 min to fully separate the supernatant and the pellet.

[0104] 4. Use 5 column volumes of binding buffer (50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0) to balance the Ni-NTA (Hangzhou New Dragon, catalog number: NRPB58S-Y15) affinity column. Then add the protein supernatant and incubate at 4°C for 0.5h-1h to allow the target recombinant protein to fully bind to the column material. Then use 200ml of washing buffer (200mM imidazole, 50mM Tris-HCl, 300mM NaCl, pH 8.0) containing 200mM imidazole to elute the target protein. Finally, add an appropriate amount of TEV protease with a His tag, incubate at 4°C for 16h, and collect the flow-through, which is the target collagen protein with the carrier protein removed. The obtained product is dialyzed overnight.

[0105] Example 2 Purification of recombinant type III collagen and removal of endotoxin

[0106] 1. According to steps 1-4 in Example 1, the target collagen protein with the carrier protein removed was obtained and dialyzed into solution A (400 mM NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5) of an anion exchange column.

[0107] 2. Use 5 column volumes of solution A (400mM NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5) to balance the anion exchange column (Q NUPharose HP, 5ml, Hangzhou New Dragon). Then load the dialyzed protein onto the column and collect the protein peak that passes through, which is the purified protein. The obtained product is dialyzed overnight.

[0108] 3. Elute the anion exchange column with 5 column volumes of solution B (1.0 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.5) to wash the impurity proteins and endotoxins off the column.

[0109] 4. The purity of the obtained recombinant protein was tested by SDS-PAGE. The results are as follows Figure 2The specific process is as follows: take 30 μL of the purified protein solution, add 10 μL of 4× protein loading buffer (200 mM Tris-HCl (pH 6.8), 8% SDS, 0.08% bromophenol blue, 40% glycerol, 50 mM EDTA, 4% β-mercaptoethanol), boil in 100°C boiling water for 10 min, then add 10 μL per well to the SDS-PAGE protein gel, run at 80 V for 2 h, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 30 min, and then use protein decolorization solution (10% acetic acid, 5% ethanol) for decolorization.

[0110] 5. The recombinant type III collagen obtained after purification and removal of endotoxin and bovine collagen were used as controls, and the activity of the recombinant type III collagen was measured by detecting the adhesion of the two proteins to cells.

[0111] Figure 2 The purification of recombinant type III collagen is shown. The purity of type III collagen is about 95% after Ni column and anion exchange.

[0112] Figure 3 The results show that recombinant type III collagen has a significant cell adhesion effect compared to the negative control (D-PBS solution), and its cell adhesion effect is consistent with that of bovine collagen.

[0113] Example 3 Comparison of cell adhesion activity of recombinant type III collagen composed of different sequence connection orders

[0114] 1. According to steps 1-4 in Example 1 and steps 1-3 in Example 2, recombinant collagens with amino acid sequences of Col3-9 to Col3-13 were obtained, and the obtained recombinant collagens Col3-9 to Col3-13 were purified and endotoxins were removed.

[0115] The three sequences of SEQ ID No.2 (GAPGER), SEQ ID No.3 (GERGAPGFRGPAGPNGIPGEKGPAGERGAP) and SEQ ID No.4 (GVMGFP) were connected in another order to form a sequence different from SEQ ID No.1, with 14 repeats, forming a recombinant type III collagen different from SEQ ID No.5:

[0116] (1) SEQ ID No. 2-SEQ ID No. 4-SEQ ID No. 3 are connected to form SEQ ID No. 9: GAPGERGVMGFPGERGAPGFRGPAGPNGIPGEKGPAGERGAP, which has 14 repeating sequences, to form recombinant type III collagen Col3-9;

[0117] (2) According to the connection order of SEQ ID No.4-SEQ ID No.3-SEQ ID No.2, SEQ ID No.10 is formed: GVMGFPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGAPGER, the number of which is 14 repeats, forming recombinant type III collagen Col3-10;

[0118] (3) According to the connection order of SEQ ID No.3-SEQ ID No.4-SEQ ID No.2, SEQ ID No.11: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGER, the number of its repeating sequences is 14, forming recombinant type III collagen Col3-11;

[0119] (4) According to the connection order of SEQ ID No.4-SEQ ID No.2-SEQ ID No.3, SEQ ID No.12 is formed: GVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAP, the number of which is 14 repeating sequences, forming recombinant type III collagen Col3-12;

[0120] (5) According to the connection order of SEQ ID No.3-SEQ ID No.2-SEQ ID No.4, SEQ ID No.13: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGAPGERGVMGFP, the number of its repeated sequences is 14, forming recombinant type III collagen Col3-13.

[0121] 2. The recombinant type III collagen obtained after purification and removal of endotoxin was used as a control with Col3-9 to Col3-13. By detecting the adhesion results of the six proteins to cells, the activity of recombinant type III collagen with different sequence connection orders was compared and analyzed.

[0122] Figure 4A-4CThe cell adhesion activity of recombinant type III collagen with different sequence connection orders is shown. Compared with the recombinant collagens Col3-9 to Col3-13 obtained by connecting the other five sequences, the recombinant type III collagen (SEQ ID No. 5) of the present application has a relatively more significant adhesion effect on cells, proving that the recombinant type III collagen formed according to the sequence connection order of the present application has better cell adhesion activity.

[0123] Example 4 Study on the thermal stability of recombinant type III collagen at 37°C

[0124] 1. According to Example 2, purified recombinant type III collagen was obtained, and then dialyzed with physiological saline (0.9%). The dialyzed protein solution was collected and sterilized by filtration with a 0.22 μM filter membrane. Two recombinant type III collagen products of a listed brand were selected as controls and sterilized by filtration with a 0.22 μM filter membrane.

[0125] 2. The dialyzed and filter-sterilized recombinant type III collagen and two control proteins were dissolved in water for injection at a concentration of 1 mg / mL. The collagen solution was then dispensed into 1.5 mL endotoxin-free Eppendorf tubes, with 1 mL of solution in each tube. All solutions were placed in a 37°C constant temperature incubator. One tube of collagen solution was taken out every 24 hours, and the degradation of collagen was detected by SDS-PAGE (see the method in Example 1). Figure 5 A shows the electrophoresis images of recombinant type III collagen at 24h, 48h, 72h and 96h, and 5B shows the electrophoresis images of recombinant type III collagen and two control proteins at 96h. Type III collagen can still maintain good structural stability after 96h at 37℃, with only a small amount of degradation, while the two control proteins are basically completely degraded after 96h, proving that recombinant type III collagen has good structural stability under human body temperature and is better than some recombinant type III collagen products that have been marketed.

[0126] Example 5 Endotoxin detection of recombinant type III collagen

[0127] 1. According to Example 2, prepare purified recombinant type III collagen.

[0128] 2. Prepare reagents, including horseshoe crab reagent (Zhanjiang Andus Biological Co., Ltd., λ = 0.015EU / mL) and diluent I (Zhanjiang Andus Biological Co., Ltd.).

[0129] 3. Dilute the endotoxin standard to E1, E0.5, E0.25, E0.125, and E0.0625. Use diluent I to dilute the sample 5, 8, 16, 32, 50, 100, 200, 300, and 400 times, and dilute the rest with detection water, so that the final concentration of the sample is 10, 16, 32, 64, 100, 200, 400, 600, and 800 times, respectively.

[0130] 4. Add endotoxin standards and diluted protein samples to the horseshoe crab reagent for observation. Compare the results with the negative and positive controls. Calculate the sample endotoxin.

[0131] After type III collagen is purified by Ni column and anion exchange column, the endotoxin content can be controlled below 0.5EU / mg.

[0132] Example 6 Structural prediction of recombinant type III collagen using AlphaFold

[0133] 1. Sequence input:

[0134] The amino acid sequence of SEQ ID No.5 was imported into the AlphaFold model.

[0135] 2. Multiple Sequence Alignment (MSA):

[0136] Multiple sequence alignment of SEQ ID No. 5 protein sequence with homologous sequences from different organisms. MSA provides valuable evolutionary information and helps improve the accuracy of structure prediction.

[0137] 3. AlphaFold prediction:

[0138] The AlphaFold deep learning model Q, which has been trained on a large number of protein structure datasets, is used to predict the three-dimensional structure of the target protein. AlphaFold uses a neural network architecture, takes MSA and target sequence as input, and predicts the distance between residue pairs in the protein.

[0139] 4. Distance matrix generation:

[0140] The predicted inter-residue distances are used to generate a distance matrix representing the pairwise distances between all amino acid residues in the protein.

[0141] 5.Structure prediction:

[0142] The predicted distance matrix is ​​used to predict the three-dimensional coordinates of the protein. This step involves solving a distance geometry problem to determine the atomic spatial arrangement that best fits the predicted distances.

[0143] 6. Refinement:

[0144] The initial predicted structure may be refined to improve its accuracy. This may involve various techniques such as molecular dynamics simulations or energy minimization to optimize the structure and eliminate any steric clashes.

[0145] 7. Structural evaluation:

[0146] Evaluate the quality of predicted protein structures. There are a variety of metrics and tools available to assess the accuracy and reliability of predicted structures, including root mean square deviation (RMSD) and MoIProbity score.

[0147] 8. Visualization and Analysis:

[0148] The predicted three-dimensional protein structure was visualized and analyzed using molecular visualization software. Figure 6 shown. Figure 6 The purpose is to display the three-dimensional structure of the recombinant collagen predicted by Alphfold, so as to visualize the structure of the recombinant collagen of the present application.

[0149] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0150] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. Recombinant humanized type III collagen, characterized in that: The recombinant humanized type III collagen includes the amino acid fragment shown in SEQ ID No.

5.

2. A nucleic acid molecule, characterized in that It comprises a nucleic acid encoding the recombinant humanized type III collagen according to claim 1.

3. An expression vector, characterized in that It comprises the nucleic acid molecule according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector contains a nucleic acid encoding the amino acid fragment shown in SEQ ID NO.

7.

5. A host cell, characterized in that It expresses the recombinant humanized type III collagen described in claim 1, contains the nucleic acid molecule described in claim 2, or contains the expression vector described in claim 3 or 4.

6. The method for preparing the recombinant humanized type III collagen according to claim 1, characterized in that: The method comprises culturing the host cell according to claim 5 and isolating the recombinant humanized type III collagen from the obtained culture product.

7. The preparation method according to claim 6, characterized in that: The step of enzymatic protein cleavage is also included.

8. A product, characterized in that It comprises the recombinant humanized type III collagen according to claim 1.

9. Use of the recombinant humanized type III collagen according to claim 1 in the preparation of a product for promoting cell adhesion.

10. Use of the recombinant humanized type III collagen according to claim 1 in preparing tissue repair materials.

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