Recombinant human type iii collagen for promoting angiogenesis and its preparation method and application

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant human collagen type III for promoting angiogenesis and a preparation method and application thereof. The recombinant human collagen type III for promoting angiogenesis has an amino acid sequence as shown in SEQ ID NO. 1. In the application, nine different sites of full-length natural collagen type III are replaced by GFPGER sequences from collagen type I which specifically bind to integrin sites, the function of promoting angiogenesis of the full-length natural collagen type III is enhanced without changing the total length of the amino acids, meanwhile, four natural integrin sites with low adhesion are replaced, so that the recombinant collagen type III has higher adhesion to further promote angiogenesis. Through the modification mode, the angiogenesis rate is increased by more than 37%, and the translated protein has moderate molecular weight and is easy to prepare.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant human type III collagen for promoting blood vessel formation and a preparation method and application thereof. BACKGROUND

[0002] Collagen is one of the most important and abundant proteins in mammals, and is a structural protein found in the skin, connective tissue and bone of the human body and other tissues. The content of collagen in the human body is about 30% of the total protein. Collagen is a structural protein, which is the main component of the extracellular matrix. Type III collagen is twisted into a triple helix by three polypeptide chains curling to the right. Primary structure analysis shows that the long segment sequence of the polypeptide chain is repeated by Gly-X-Y amino acid sequence. Among them, X is usually proline, and Y is usually hydroxyproline and hydroxylysine, which are rarely seen in other proteins.

[0003] Collagen has low immunogenicity, and has functions of promoting tissue repair, hemostasis and the like, and has been widely applied to the fields of food, cosmetics, biomedical materials, drugs and the like. At present, collagen is mainly extracted from animal tissues, however, materials derived from animal tissues all have the risk of viral infection, such as mad cow disease and the like. Meanwhile, natural collagen is widely present in skin tissue and blood vessels, and is up-regulated in the growth and wound healing process of the organism, but the angiogenesis rate of natural collagen is low. SUMMARY

[0004] The application aims to provide a recombinant human type III collagen for promoting angiogenesis and a preparation method and application thereof, and solves the problem of low angiogenesis rate of natural collagen.

[0005] The technical scheme adopted by the application 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 a cardiovascular stent material or an artificial blood vessel.

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

[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 in SEQ ID NO. 2.

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

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

[0012] A method for preparing the recombinant human type III collagen by using the host cell, wherein the host cell is cultured in a culture medium, and after inducing the expression of the recombinant human type III collagen, the recombinant human type III collagen is purified to obtain the recombinant human type III collagen.

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

[0014] The recombinant human type III collagen has higher adhesion and further promotes angiogenesis.

[0015] The recombinant collagen of the application is codon-optimized to better adapt to the host bacteria.

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

[0017] The recombinant collagen prepared by the application is expressed by Pichia pastoris engineering bacteria, and the protein has no endotoxin hidden danger, and the protein does not carry a histidine marker, and can be purified by molecular sieves, so that the target protein can be directly obtained, and the histidine marker sequence does not need to be additionally removed.

[0018] The genetically recombinant collagen prepared by the method of the application can significantly promote angiogenesis, and is applied to cardiovascular diseases, wound healing and skin repair, and does not cause immune rejection, and can be widely applied to the fields of medical materials, cosmetics and the like.

[0019] The preparation method of the recombinant human type III collagen of the application is suitable for industrial large-scale production, and the prepared product has no animal-derived infectious source, so that the biological safety is higher. BRIEF DESCRIPTION OF DRAWINGS

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

[0021] Figure 2 It is a result of the influence of different concentrations of the recombinant human type III collagen on cell proliferation.

[0022] Figure 3 The angiogenesis graph of Example 3 is shown from left to right as BSA, type III natural collagen, type III recombinant collagen. DETAILED DESCRIPTION

[0023] In order for those skilled in the art to better understand the technical solutions of the present application, the present application is further described below in conjunction with specific examples and drawings.

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

[0025] The inventive 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, which occupies most of the connective tissue and provides the necessary physical framework for cell components. Collagen, as the main component of ECM, is recognized by receptors such as integrins, discoid domain receptors, and multi-ligand proteoglycans, and plays an important functional role in controlling key cell responses such as adhesion, migration, proliferation, differentiation, and survival.

[0026] Since there is a lot of type III collagen in blood vessels, the inventors replaced 9 different sites of full-length natural type III collagen with GFPGER sequences from type I collagen that specifically bind to integrin sites, enhancing its pro-angiogenic function without changing the total length of the natural full-length amino acid, and also replacing 4 natural integrin sites with lower adhesion rates, so that the recombinant type III collagen has higher adhesion to further promote angiogenesis.

[0027] The present application aims to provide a recombinant human type III recombinant collagen with the ability to promote blood vessel formation, which can effectively promote blood vessel formation and be applied in cardiovascular diseases, wound healing, and skin repair, and will not cause immune rejection, and can be widely used in medical materials, cosmetics, and other fields.

[0028] The present application provides a type III recombinant human collagen with the ability to promote angiogenesis, and the amino acid sequence is shown in SEQ ID NO. 1.

[0029] SEQ ID NO. 1:

[0030]

[0031] The present application provides a human type III recombinant collagen with pro-angiogenic ability, which is codon-optimized for host cell expression, has a signal peptide cleavage site added at each end during design, and Eco R I and Not I enzyme cleavage sites to facilitate later genetic manipulation. After the above optimization, the nucleotide sequence is shown in SEQ ID NO. 2.

[0032] SEQ ID NO. 2:

[0033]

[0034] The present application provides an expression vector comprising the above nucleic acid molecule. The vector can contain regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host, e.g., bacterial, fungal, plant or animal, into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA-based or RNA-based. In a specific embodiment, the expression vector is pPIC9k, the nucleotide sequence of which is shown as SEQ ID NO. 3.

[0035] SEQ ID NO. 3:

[0036]

[0037] The present application provides a host cell comprising the above-mentioned nucleic acid molecule. The host cell refers to a cell into which a foreign nucleic acid has been introduced, including the progeny of such a cell. The host cell includes transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages.

[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 application provides a method for preparing any one of the above-mentioned recombinant collagens, comprising the following steps:

[0041] The expression is carried out using the above-mentioned host cell, and then separation and purification are carried out.

[0042] The expression of the host cell refers to the culture of the host cell, and 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, and after obtaining the Pichia pastoris genetically engineered bacteria, the specific culture conditions are as follows: inoculate the Pichia pastoris genetically engineered bacteria into YPD culture medium, and culture at 30°C and 220 rpm for 22h-24h until the OD600 is 18-20 as the upper tank seed liquid, inoculate the seed liquid at a volume of 10% after expansion into an initial volume of 5L NBS 415 fermentation tank, the culture temperature is 28°C-30°C, the pH is 5.0-6.0, the dissolved oxygen is controlled at 20%-30%, and after the glycerol is consumed, the induction culture is started.

[0044] As for the expression mode, the present application does not make any limitation, which can be determined as needed, for example, expression as induced expression, and as for the induced expression, the inducer is methanol.

[0045] In a specific embodiment, methanol is added for induction culture, the induction stage temperature is 28°C, the pH is 5.0, and the induction is carried out for 48h before tank discharge.

[0046] As for the method of separation and purification, the present application does not make any limitation, which can be determined as needed, for example, salt precipitation method, ultrafiltration method, affinity chromatography method, and gel filtration chromatography method can be used.

[0047] The present application also provides the recombinant human type III collagen protein, the recombinant collagen protein encoded by the nucleic acid molecule, the recombinant human type III collagen protein expressed by the expression vector, or the recombinant human type III collagen protein produced by the host cell, which has the abilities of promoting angiogenesis and wound healing.

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

[0049] The recombinant collagen protein gene of the present application is chemically synthesized, and the nucleotide sequence is shown in SEQ ID NO. 2. The 5' end and 3' end are added with Eco RI and Not I recognition site and signal peptide recognition site, respectively, and the linearized by restriction enzyme Sac I, and then cloned into the expression vector pPIC9K to obtain the pPIC9K-ROL(III) cloning plasmid. The obtained pPIC9K-ROL(III) cloning plasmid is linearized by electroporation and transformed into GS115 to obtain GS115-pPIC9K-ROL(III), and the Pichia pastoris genetic engineering bacteria are obtained by culturing at 30°C for 72h.

[0050] The obtained Pichia pastoris genetic engineering bacteria are inoculated into YPD culture medium and cultured to OD 600 When the OD is 19.88, the inoculation amount is 10%, and the initial volume of the NBS 415 fermentation tank is 5L. The culture temperature is 30°C, the pH is 5.5, and the dissolved oxygen is controlled at 20%. After the glycerol is consumed, the glycerol feeding culture is started. When the wet weight of the bacteria reaches more than 190g / L, the methanol is added at a flow rate of 80mL / h for induction culture. The induction stage temperature is 28°C, the pH is 5.0, and the induction is performed for 48h. The supernatant is collected by centrifugation.

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

[0052] 1. The centrifuged supernatant is ultrafiltrated to 50% of the initial volume, 5 times the volume of pure water is added, and then ultrafiltrated and concentrated to 5% of the initial volume.

[0053] 2. The concentrated supernatant is added with saturated ammonium sulfate, and the addition amount is 60% of the volume of the concentrated supernatant. After stirring at room temperature for 30min, the precipitate is collected by centrifugation at 9000rpm for 10min. The obtained precipitate is dissolved in 500mL of 0.05M PBS with pH of 7.0, and then filtered through a 0.22μm filter membrane.

[0054] 3. Prepare the equilibrium buffer according to the isoelectric point of the protein: 20 mmol / L sodium phosphate buffer, pH 6.0, noted as A; and eluent prepared by mixing 20 mmol / L sodium phosphate and 1.0 mol / L NaCl, pH 6.0, noted as B. Prepare the sample solution by diluting the PBS protein solution obtained in the previous step with A at 10:1, filter and load onto a 25 mL CM-Sepharose cation exchange chromatography column, which has been equilibrated with the equilibrium buffer. After loading, wash the column with A for 2 column volumes, and then elute with a gradient of 70% A and 30% B at a flow rate of 2 mL / min. Collect the eluted fractions and test them by SDS-PAGE. The results are shown in Fig. 2, where M represents the marker, lane 1 is the sample solution, lane 2 is the sample flow-through, and lane 3 is the purified protein, i.e., recombinant GS115-pPIC9K-ROL(III) protein. Figure 1

[0055] 4. According to the molecular weight distribution of the protein obtained after ion exchange chromatography, select a Sephadex 200 gel column for further purification of the target protein. The AKTA operation process is as follows: first, wash with the equilibrium buffer, i.e., 0.01 mol / L PBS and 0.05 mol / L NaCl, until the baseline is stable, and then load the recombinant GS115-pPIC9K-ROL(III) protein fraction obtained in the previous step onto a gel filtration chromatography column packed with Superdex 200, elute with the eluent, set the flow rate to 10 mL / min, and set the ultraviolet detection wavelength to 215 nm. Finally, collect the target protein fraction after detecting it by SDS-PAGE electrophoresis.

[0056] 5. Ultrafiltration desalting: desalting with a G25 desalting column, i.e., use 25 mL of G25 packing, and the operation process is similar to the gel filtration chromatography step, load 6.5 mL each time, collect about 8 mL, and complete the desalting after 10 min of loading.

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

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

[0059] 1. Cell proliferation experiment

[0060] ​In vitro cell proliferation experiment model was used to evaluate the cell activity induced by recombinant GS115-pPIC9K-ROL(III) protein. In short, HUVEC cells in logarithmic growth phase were inoculated 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 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, and then added to the cells for incubation for 24 h. The old culture medium was discarded. In the control group, 100 μL of fresh complete culture medium was added, while in the experimental group, 100 μL of the same volume of recombinant GS115-pPIC9K-ROL(III) protein solution was added. Subsequently, cell proliferation was verified using the cck8 method, and the absorbance was measured at 450 nm. The cell proliferation rate was calculated as follows: Figure 2 .

[0061] 2. Pro-angiogenic experiment

[0062] Preparation of Matrigel

[0063] The Matrigel stored at -20°C was buried in crushed ice in a 4°C refrigerator the day before the experiment, allowing the Matrigel to slowly thaw overnight. Before the experiment, the Matrigel was placed in an ice box, and the 48-well plate and 1 mL gun tip were pre-cooled on ice. The Matrigel was mixed with the pre-cooled gun tip.

[0064] Laying Matrigel

[0065] Pre-cooled 1.5 mL centrifuge tubes were prepared for diluting the Matrigel. The Matrigel was diluted at a volume ratio of 1:1 with DMEM medium. After dilution, 80 μL of the diluted Matrigel was added to each well of the 48-well plate, and the addition was done vertically to avoid air bubbles. The Matrigel was allowed to solidify in a 37°C incubator for 1 hour.

[0066] Laying cells

[0067] HUVEC cells in good condition at passage 5 were used. The cells were digested, resuspended in DMEM medium containing 10% serum, and counted. About 50,000 cells were added to each well of the 48-well plate in a volume of 100 μL, with three repeats per group. BSA, native type III collagen, and purified recombinant GS115-pPIC9K-ROL(III) protein were sequentially dissolved in DMEM medium at a final concentration of 3 mg / mL, and were sequentially referred to as the negative control group, the positive control group, and the experimental group, with a volume of 200 μL added to each well of the 48-well plate. The 48-well plate was placed in a 37°C incubator for culture, and blood vessel formation was observed after 12 hours.

[0068] Quantitative analysis

[0069] The blood vessel network was quantitatively counted using an Angiogenesis Analyzer plug-in in Image J, and the angiogenesis rate statistics are shown in Table 1, and the angiogenesis graph is shown in Figure 3 .

[0070] Table 1 Angiogenesis rate statistics table

[0071]

[0072] The results show that the recombinant human Ш recombinant collagen of the application can significantly promote HUVEC cell angiogenesis, the pro-angiogenesis ability is higher than that of natural collagen, and is significantly higher than that of BSA, has good cell compatibility, and shows good promotion, which is consistent with the light microscope observation results.

[0073] The technical features of the above-described embodiments can be combined arbitrarily, and to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered as the scope of the description.

[0074] The above-described embodiments only express several implementation manners of the application, the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent application. It should be pointed out that for ordinary skilled persons in the art, on the premise of not departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application.

Claims

1. A recombinant human collagen type III which promotes angiogenesis, characterized in that, The amino acid sequence is shown as SEQ ID NO.

1.

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

2.

3. The recombinant human collagen type III according to claim 1, wherein, The recombinant human collagen type III is used for preparing cardiovascular stent material or artificial blood vessel.

4. The recombinant human collagen type III according to claim 1, wherein, The recombinant human collagen type III is used for preparing skin tissue repair material.

5. An expression vector, characterized by, The nucleotide sequence is shown as SEQ ID NO.

2.

6. A host cell, characterized in that, The expression vector is shown as SEQ ID NO.

5.

7. The host cell of claim 6, wherein, The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

8. A method for producing recombinant human collagen type III using the host cell of claim 6, characterized by, The host cell is cultured in a culture medium, and after the expression of the recombinant human collagen type III is induced, the recombinant human collagen type III is obtained through purification.

9. The method of claim 8, wherein, The purification method is any one of salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.

Images