Recombinant human III-type collagen for promoting angiogenesis as well as preparation method and application of recombinant human III-type collagen

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant human III-type collagen for promoting angiogenesis as well as a preparation method and application of the recombinant human III-type collagen. The recombinant human III-type collagen for promoting angiogenesis has an amino acid sequence as shown in SEQ ID NO. 1. Nine different sites of the full-length natural III-type collagen are replaced by GFOGER sequences which are from the I-type collagen and are specifically combined with integrin sites, the angiogenesis promoting function of the collagen is enhanced under the condition that the total length of natural full-length amino acid is not changed, and meanwhile, four natural integrin sites with low adhesion rate are also replaced; the recombinant III-type collagen has high adhesive force, so that angiogenesis is further promoted. By means of the transformation mode, the angiogenesis rate is increased by 37% or above, the molecular weight of the translated protein is moderate, and the translated protein is easy to prepare.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant human type III collagen protein that promotes angiogenesis, and a preparation method and application thereof. Background Art

[0002] Collagen is one of the most important and abundant proteins in mammals. It is a structural protein found in human skin, connective tissue, bones and other tissues. The content of collagen in the human body is about 30% of the total protein. Collagen is a structural protein and the main component of the extracellular matrix. Type III collagen is composed of three peptide chains curled to the right to form a triple helix. Primary structure analysis shows that the long segment sequence of its polypeptide chain is composed of repeated Gly-XY amino acid sequences. Among them, X is usually proline, and Y is usually hydroxyproline and hydroxylysine. The latter two amino acids are rarely found in other proteins.

[0003] Collagen has low immunogenicity and has the functions of promoting tissue repair and hemostasis. It is now widely used in food, cosmetics, biomedical materials, pharmaceuticals and other fields. At present, collagen is mainly extracted from animal tissues. However, materials derived from animal tissues have the risk of viral infection, such as mad cow disease. At the same time, natural collagen is widely present in skin tissues and blood vessels, and its expression is upregulated during the growth and wound healing of organisms, but the angiogenesis rate of natural collagen is low. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant human type III collagen protein that promotes angiogenesis, and a preparation method and application thereof, thereby solving the problem that the angiogenesis promoting rate of natural collagen is low.

[0005] The technical solution adopted by the present invention is a recombinant human type III collagen for promoting angiogenesis, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The recombinant human type III collagen is used for preparing cardiovascular stent materials or artificial blood vessels.

[0007] The recombinant human type III collagen is used for preparing skin tissue repair materials.

[0008] The recombinant human type III collagen is used for preparing a preparation for treating cardiovascular diseases.

[0009] An expression vector comprises the nucleotide sequence shown as SEQ ID NO.2.

[0010] A host cell comprises the above expression vector.

[0011] The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

[0012] A method for preparing recombinant human type III collagen using the host cell comprises culturing the host cell in a culture medium, inducing the expression of the recombinant human type III collagen, and then purifying the recombinant human type III collagen to obtain the recombinant human type III collagen.

[0013] The purification method is selected from any one of salting out, ultrafiltration, affinity chromatography and gel filtration chromatography

[0014] The beneficial effects of the present invention are as follows: the present invention replaces 9 different sites of full-length natural type III collagen with GFOGER sequences from type I collagen that specifically bind to integrin sites, thereby enhancing its angiogenesis-promoting function without changing the total length of the natural full-length amino acids, and at the same time replaces 4 natural integrin sites with low adhesion rates, so that the recombinant type III collagen has higher adhesion and further promotes angiogenesis. This modification method will increase the angiogenesis rate by more than 37%, and the translated protein has a moderate molecular weight and is easy to prepare.

[0015] The recombinant collagen of the present invention can be better adapted to the host bacteria by codon optimization.

[0016] The recombinant collagen of the present invention removes the full-length chain sequence of the C-terminus and the N-terminus of the collagen coding region, thereby effectively avoiding a series of antigen immune reactions.

[0017] The recombinant collagen prepared by the present invention is expressed by Pichia pastoris engineering bacteria, has no hidden danger of endotoxin, and does not carry a histidine tag, can be purified by molecular sieve, and finally the target protein can be directly obtained without the need for additional removal of the histidine tag sequence.

[0018] The recombinant collagen prepared by the method of the present invention can significantly promote angiogenesis, and is applied to cardiovascular diseases, wound healing and skin repair, and will not cause immune rejection, and can be widely used in the fields of medical materials, cosmetics, etc.

[0019] The method for preparing recombinant human type III collagen of the present invention is suitable for industrial large-scale production, and the prepared product has no infection source of animal origin, so it has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the purification of recombinant human type III collagen.

[0021] Figure 2 The results show the effects of different concentrations of recombinant human type III collagen on cell proliferation.

[0022] Figure 3 This is a diagram of angiogenesis in Example 3, from left to right: BSA, type III natural collagen, and type III recombinant collagen. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0024] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0025] The invention concept is as follows: Angiogenesis is related to cell migration and proliferation, and cell adhesion is a prerequisite for angiogenesis. The cytoplasmic matrix, ECM, is a non-cellular component present in all tissues and organs, mainly secreted by fibroblasts. It occupies most of the connective tissue area and provides the necessary physical framework for cell assemblies. Collagen, as the main component of ECM, is recognized by receptors such as integrins, discoidin domain receptors and syndecans, and thus plays an important functional role in controlling key cell responses such as adhesion, migration, proliferation, differentiation and survival.

[0026] Since there is a large amount of type III collagen in blood vessels, the inventors replaced 9 different sites of full-length natural type III collagen with the GFOGER sequence from type I collagen that specifically binds to the integrin site, thereby enhancing its angiogenic function without changing the total length of the natural full-length amino acids. At the same time, 4 natural integrin sites with lower adhesion rates were replaced, so that the recombinant type III collagen has higher adhesion and further promotes angiogenesis.

[0027] The purpose of the present invention is to provide a recombinant human type II recombinant collagen with the ability to promote angiogenesis. The recombinant collagen that promotes angiogenesis can effectively promote angiogenesis, can be applied to cardiovascular diseases, wound healing and skin repair, and will not cause immune rejection. It can be widely used in medical materials, cosmetics and other fields.

[0028] The present invention provides a type III recombinant human collagen protein capable of promoting angiogenesis, and the amino acid sequence thereof is shown as SEQ ID NO.1.

[0029] SEQ ID NO.1:

[0030]

[0031] The present invention provides a human type III recombinant collagen protein with the ability to promote angiogenesis, wherein codon optimization is performed for host cell expression, and signal peptide cleavage sites and EcoR I and Not I restriction sites are added to both ends during the design process to facilitate later gene manipulation. After the above optimization, the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0032] SEQ ID NO.2:

[0033]

[0034] The present invention provides an expression vector comprising the above nucleic acid molecule. The vector may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host into which the vector is to be introduced, for example, bacteria, fungi, plants or animals, taking into account whether the vector is based on DNA or RNA. In a specific embodiment, the expression vector is pPIC9k, and its nucleotide sequence is shown in SEQ ID NO.3.

[0035] SEQ ID NO.3:

[0036]

[0037] The present invention provides a host cell comprising the above-mentioned nucleic acid molecule. The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the offspring of such a cell. Host cells include transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, regardless of the number of passages. Offspring may not be completely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0038] In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0039] In a specific embodiment, the host cell is Pichia pastoris GS115.

[0040] The present invention provides a method for preparing any of the aforementioned recombinant collagen, comprising the following steps:

[0041] The above-mentioned host cells are used for expression and then separated and purified to obtain the product.

[0042] The term "expressing a host cell" refers to culturing the host cell. The culture medium and culture conditions are well known to those skilled in the art.

[0043] In a specific embodiment, the host cell is Pichia pastoris. After obtaining the genetically engineered Pichia pastoris, the specific culture conditions are as follows: the genetically engineered Pichia pastoris is inoculated into a YPD medium and cultured at 30°C and 220rpm for 22h to 24h until the OD600 is 18 to 20 as the seed liquid for the upper tank. After the seed liquid is expanded, it is inoculated into an NBS 415 fermenter with an initial volume of 5L at a 10% inoculum amount by volume. The culture temperature is 28°C to 30°C, the pH is 5.0 to 6.0, and the dissolved oxygen is controlled at 20% to 30%. When the glycerol is exhausted, glycerol fed culture is started, and when the wet weight of the bacteria reaches 180g / L or more, induction culture is started.

[0044] The present invention does not impose any limitation on the expression mode, which can be confirmed as needed, for example, the expression is induced expression, and the inducer for induced expression is methanol.

[0045] In a specific embodiment, methanol is added for induction culture, the temperature during the induction stage is 28° C., the pH is 5.0, and the culture is released after 48 hours of induction.

[0046] The present application does not impose any limitation on the separation and purification method, which can be determined according to the method, for example, salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method can be used.

[0047] The present invention also provides the above-mentioned recombinant human Ш recombinant collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant human Ш recombinant collagen expressed by the above-mentioned expression vector, or the recombinant human Ш recombinant collagen produced by the above-mentioned host cell, which has the ability to promote angiogenesis and wound healing.

[0048] Example 1: Expression of recombinant human type III collagen

[0049] The recombinant collagen gene of the present invention was chemically synthesized, and the nucleotide sequence was shown in SEQ ID NO. 2. During the synthesis, EcoR I and Not I recognition sites and signal peptide recognition sites were added to the 5' end and 3' end, respectively, and the gene was linearized by restriction endonuclease Sac I and cloned into the expression vector pPIC9K to obtain the pPIC9K-ROL (III) cloning plasmid, and Pichia pastoris GS115 was used as the expression host bacteria. The obtained pPIC9K-ROL (III) cloning plasmid was linearized by electroporation and transformed into GS115 to obtain GS115-pPIC9K-ROL (III), and the Pichia pastoris genetic engineering bacteria were obtained by culturing at 30° C. for 72 hours.

[0050] The genetically engineered Pichia pastoris obtained above was inoculated into YPD medium and cultured until OD 600 When the pH value was 19.88, the fermentation tank was inoculated with 10% of the inoculation amount into an NBS 415 fermenter with an initial volume of 5 L. The culture temperature was 30°C, the pH was 5.5, and the dissolved oxygen was controlled at 20%. When the glycerol was exhausted, the glycerol fed culture was started. When the wet weight of the bacteria reached more than 190 g / L, methanol was added at a flow rate of 80 mL / h for induction culture. The temperature in the induction stage was 28°C and the pH was 5.0. The fermentation tank was opened after 48 hours of induction and the supernatant was collected by centrifugation.

[0051] Example 2: Purification of recombinant human type III collagen

[0052] 1. When the supernatant collected by centrifugation is ultrafiltered to 50% of the initial volume, 5 times the volume of pure water is added and then ultrafiltered to concentrate to 5% of the initial volume.

[0053] 2. Add saturated ammonium sulfate to the concentrated supernatant in an amount of 60% of the volume of the concentrated supernatant, stir at room temperature for 30 minutes, centrifuge at 9000 rpm for 10 minutes, collect the precipitate, dissolve the precipitate in 500 mL of 0.05 M PBS, pH 7.0, and filter through a 0.22 μm filter membrane.

[0054] 3. Prepare the equilibrium buffer according to the isoelectric point of the protein: that is, 20mmol / L, pH6.0 sodium phosphate buffer, recorded as liquid A; prepare the eluent of pH6.0 by mixing sodium phosphate with a molar mass of 20mmol / L and 1.0mol / L NaCl, recorded as liquid B. Use liquid A to dilute the PBS protein solution obtained in the previous step at a ratio of 10:1 to prepare the loading solution, and then load it onto a 25mL CM-Sepharose cation exchange chromatography column after filtration. Before loading, balance the column with equilibrium buffer. After loading, rinse 2 column volumes with liquid A, and then use 70% liquid A and 30% liquid B for gradient elution at a flow rate of 2mL / min. Collect the eluted components and detect them by SDS-PAGE. The results are as follows Figure 1 As shown, M: marker, lane 1 is the loading solution, lane 2 is the loading effluent, and lane 3 is the purified protein, namely the recombinant GS115-pPIC9K-ROL(III) protein.

[0055] 4. According to the distribution range of protein molecular weight obtained after ion exchange chromatography, select Sephadex 200 gel column to further purify the target protein. AKTA operation process: first use equilibrium buffer, i.e. 0.01mol / L PBS, 0.05mol / L NaCl, to rinse until the baseline is stable, and then load the recombinant GS115-pPIC9K-ROL (III) protein component eluted from the ion exchange column obtained in the previous step onto a gel filtration chromatography column filled with Superdex 200, eluted with eluent, the flow rate is set to 10mL / min, and the ultraviolet detection wavelength is 215nm. Finally, collect the outflowing target protein component after SDS-PAGE electrophoresis detection.

[0056] 5. Ultrafiltration desalting: Desalting by G25 desalting column, that is, using 25mL of G25 filler. The operation process is similar to the gel filtration chromatography step. Load 6.5mL each time and collect about 8mL. Desalting can be completed 10 minutes after loading.

[0057] 6. Concentrate to 30% of the initial volume by ultrafiltration, then pre-freeze in a -20°C refrigerator for 4 hours, then transfer to a vacuum freeze dryer for freeze-drying, collect the freeze-dried protein after 48 hours, and store the freeze-dried protein sample in a -80°C refrigerator for later use.

[0058] Example 3: Application of recombinant human type III collagen

[0059] 1. Cell proliferation assay

[0060] The in vitro cell proliferation experimental model was used to evaluate the cell activity induced by the recombinant GS115-pPIC9K-ROL(III) protein. Briefly, HUVEC cells in the logarithmic growth phase were seeded on 96-well tissue culture plates and cultured in a biochemical incubator at 37°C with a volume fraction of 5% CO2 for 1 day. The purified recombinant GS115-pPIC9K-ROL(III) protein was dissolved in DMEM culture medium and prepared into standard extracts with concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL and 5 mg / mL, filtered and sterilized, added to the cells and incubated for 24 hours, and the old culture medium was discarded. In the control group, 100 μL of fresh complete culture medium was added, while the experimental group was 100 μL of an equal volume of recombinant GS115-pPIC9K-ROL(III) protein solution. Subsequently, cell proliferation was verified using the cck8 method, and the absorbance was measured at 450 nm. The cell proliferation rate is shown in Figure 2 .

[0061] 2. Angiogenesis Experiment

[0062] Prepare Matrigel

[0063] The day before the experiment, the matrix gel stored at -20℃ was buried in crushed ice and placed in a 4℃ refrigerator to allow the matrix gel to melt slowly overnight. Before the experiment, the matrix gel was placed in an ice box, the 48-well plate and 1mL pipette tip were placed on ice for precooling, and the matrix gel was mixed with the precooled pipette tip.

[0064] Matrigel

[0065] Prepare a pre-cooled 1.5 mL centrifuge tube for diluting the matrix gel, and dilute the matrix gel with a volume ratio of 1:1 between the matrix gel and DMEM medium. After dilution, add 80 μL of the diluted matrix gel to each well of the 48-well plate, vertically adding to avoid bubbles. Place in a 37°C incubator for 1 hour to allow the matrix gel to solidify.

[0066] Cell plating

[0067] Use HUVEC cells in good condition at the 5th generation, digest the cells, resuspend the cells in DMEM medium containing 10% serum and count them. Add 100 μL of the resuspension of about 50,000 cells to each well of the above 48-well plate, and repeat three wells in each group; dissolve BSA, natural type III collagen and purified recombinant GS115-pPIC9K-ROL (III) protein in DMEM medium at a final concentration of 3 mg / mL, and record them as negative control group, positive control group and experimental group respectively, and add them to the above 48-well plate at a volume of 200 μL per well. The 48-well plate was placed in a 37°C incubator and angiogenesis was observed after 12 hours.

[0068] Quantitative analysis

[0069] The vascular network was quantitatively analyzed using the Angiogenesis Analyzer plugin in Image J. The angiogenesis rate statistics are shown in Table 1 , and the angiogenesis diagram is shown in Figure 3 .

[0070] Table 1 Statistical table of angiogenesis rate

[0071] Negative control Positive Control Experimental Group Angiogenesis rate 100％ 105％ 142％

[0072] The results show that the recombinant human collagen of the present invention can significantly promote HUVEC cell angiogenesis, and its angiogenesis-promoting ability is higher than that of natural collagen and significantly higher than that of BSA, and has good cell compatibility and exhibits a good promoting effect, which is consistent with the results of light microscopy observation.

[0073] 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.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A recombinant human type III collagen for promoting angiogenesis, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the recombinant human type III collagen as claimed in claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. The use of recombinant human type III collagen according to claim 1, characterized in that: The recombinant human type III collagen is used for preparing cardiovascular stent materials or artificial blood vessels.

4. The use of recombinant human type III collagen according to claim 1, characterized in that: The recombinant human type III collagen is used for preparing skin tissue repair materials.

5. The use of recombinant human type III collagen according to claim 1, characterized in that: The recombinant human type III collagen is used for preparing a preparation for treating cardiovascular diseases.

6. An expression vector, characterized in that: Comprising the nucleotide sequence as claimed in claim 2.

7. A host cell, characterized in that Comprising the expression vector as claimed in claim 6.

8. The host cell according to claim 7, characterized in that The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

9. A method for preparing recombinant human type III collagen using the host cell according to claim 7, characterized in that: The host cells are cultured in a culture medium, and the expression of recombinant human type III collagen is induced and then purified to obtain the recombinant human type III collagen.

10. The method according to claim 9, characterized in that The purification method is selected from any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.

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