Recombinant human type III collagen and its preparation method and application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a recombinant human type III collagen, a preparation method and an application thereof. The recombinant human type III collagen is prepared by intercepting a specific amino acid fragment of the full-length human type III collagen GAIGPSGPAGKDGESGRPGRPGERGLPGPP GIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD, repeating and optimizing the fragment, and the collagen has the advantages of good water solubility and high biological activity, and is suitable for applications related to the field of medical devices.

Description

Technical Field

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

[0002] Collagen, also known as collagen peptide, is divided into type I, type II, type III, etc. according to the order of discovery in the body. Among them, type III collagen is a triple helix structure formed by three identical α-peptide chains spirally wound together. It has a wide range of application value in maintaining the elasticity of skin / blood vessels, tissue growth and repair, etc. For example, type III collagen and type I collagen together form the basement membrane of the skin, providing strength and support for the skin, and ensuring the tension and toughness of the skin; type III collagen promotes wound healing and cell migration during the skin regeneration process; in addition, type III collagen is also used as a biomedical material to make artificial corneas, artificial heart valves, and endometrial repair.

[0003] At present, the main sources of collagen are animal tissue extraction, chemical synthesis and genetic engineering methods. Among them, heterologous collagen extracted from animal tissues has certain immunogenicity and is limited by the limited source of raw materials, which limits the development and application of animal collagen. Although chemical synthesis solves the problem of immunogenicity, the synthesis technology is relatively complex, the cost is high and there is no biological activity, so it is limited to laboratory research. Collagen prepared by genetic engineering methods has the advantages of single component, high safety, controllable production process, and more conducive to industrialization, which is currently a research hotspot. At present, the preparation of collagen by genetic engineering methods often uses Pichia pastoris and Escherichia coli for expression, but Pichia pastoris expression has defects such as long expression cycle and high culture cost; while Escherichia coli has strong reproduction ability, high culture density, low culture medium feeding cost, and is relatively economical and practical, but the biological activity of its expression product is low, such as CN118684761A discloses a recombinant type III humanized collagen, which is expressed in Escherichia coli, and the collagen concentration is 200ug / mL, and its cell activity is only about 1.3; CN115521371B discloses an Escherichia coli expression system of a recombinant humanized type III collagen, and the collagen is cultured for 24h at 1mg / mL, and its cell proliferation activity is not significantly different from that of the blank group. CN114671946B discloses that a human type III collagen fragment is repeated 5 times to obtain a recombinant human type III protein, and although there is no obvious degradation band when stored at room temperature for 12 months, its cell activity needs to be further improved.

[0004] Therefore, it is very valuable to study and develop a highly active type III collagen suitable for prokaryotic expression system, especially Escherichia coli expression. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant human type III collagen, which is expressed in an Escherichia coli system, has a small molecular weight, good water solubility and excellent biological activity; at the same time, the present invention also provides a preparation method for efficiently expressing the type III collagen and its application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides a recombinant human type III collagen, characterized in that the amino acid sequence of the recombinant human type III collagen is:

[0008] (GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD) n

[0009] The n is ≥ 2; preferably n is 3 to 10; more preferably n is 4.

[0010] In certain embodiments, the recombinant human type III collagen is characterized by having an amino acid sequence as shown in SEQ ID NO:1.

[0011] The present invention also provides a DNA sequence encoding the recombinant human type III collagen, as specifically shown in SEQ ID NO:2.

[0012] The present invention also provides a recombinant expression vector, which comprises the DNA sequence shown in SEQ ID NO:2.

[0013] The present invention also provides a recombinant expression vector, which further comprises a backbone vector, and the backbone vector is pET-32a-Kan.

[0014] The present invention also provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium comprises the recombinant expression vector described above.

[0015] Another aspect of the present invention provides a method for preparing the above-mentioned recombinant human type III collagen, comprising the following steps:

[0016] (1) Protein sequence design

[0017] Extract specific amino acid fragments of full-length human type III collagen

[0018] GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD,

[0019] Repeat 4 times to obtain the amino acid sequence of recombinant human type III collagen as shown in SEQ ID NO: 1;

[0020] (2) Construction of recombinant expression vector

[0021] Set sequence 5' - CACCACCACCATCACCATGATGATGATGACAAG-3' was connected to the 5' end of the sequence of SEQ ID NO: 2, and then inserted into the restriction sites EcoR I and Kpn I of the expression vector pET-32a-Kan to obtain a recombinant expression vector containing a recombinant human type III collagen coding gene;

[0022] (3) Construction and screening of expression strains

[0023] The recombinant expression vector described in step (2) is transferred into the host cell by heat shock method, and after overnight plating, the cell is selected and inoculated into LB resistance medium containing Kan to obtain an expression strain;

[0024] (4) Induced expression and purification

[0025] The expression strain obtained in step (3) is induced to express, the bacterial solution is collected, and purified to obtain recombinant human type III collagen.

[0026] In certain embodiments of the present invention, the host cell is Escherichia coli BL21 (DE3).

[0027] In certain embodiments of the present invention, the induction expression condition is 37° C., 1.0 mM IPTG induction for 4 h.

[0028] In certain embodiments of the present invention, the purification step is to purify the bacterial solution collected by induced expression according to the following steps in order: resuspending and disrupting the collected bacterial solution, collecting the supernatant by centrifugation, filtering, collecting the filtrate and then performing Ni affinity chromatography or cation chromatography, EK enzyme digestion, and finally anion chromatography to obtain recombinant human type III collagen.

[0029] The present invention also provides a use of the above-mentioned recombinant human type III collagen in preparing a product for promoting cell proliferation and / or promoting cell migration.

[0030] In another aspect, the present invention also provides a use of the above-mentioned recombinant human type III collagen in the field of preparing medical devices.

[0031] The medical device field described in the present invention is selected from: one or more of skin wound dressings, scar repair dressings, oral mucosa repair dressings, oral care dressings, vaginal repair dressings, vaginal care dressings, uterus repair dressings, uterus care dressings, nasal mucosa repair dressings, nasal care dressings, prostate care dressings, cavity lubricating dressings and hemorrhoid care dressings; further selected from: one or more of skin wound dressings, scar repair dressings, oral mucosa repair dressings, oral care dressings, vaginal repair dressings, vaginal care dressings, uterus repair dressings, uterus care dressings, nasal mucosa repair dressings, nasal care dressings and cavity lubricating dressings.

[0032] Beneficial effects of the present invention:

[0033] In the present invention, the inventors surprisingly discovered a recombinant human type III collagen with good water solubility and high biological activity, which is obtained by intercepting a specific amino acid sequence fragment of human type III collagen, repeating it 4 times, and undergoing protein sequence design, codon optimization, expression vector construction, induced expression and other steps. Specifically:

[0034] 1. The recombinant human type III collagen provided by the present invention has good water solubility. The induced expression bacterial solution is crushed and centrifuged, and a large amount of target protein can be harvested from the supernatant, which is conducive to subsequent industrial development.

[0035] 2. The recombinant human type III collagen provided by the present invention has excellent cell proliferation and cell migration promoting abilities. The cell proliferation promoting activity is about twice that of bovine type III collagen and the recombinant human type III collagen disclosed in CN114671946B. And in terms of cell migration, at about 6 hours, the blank group, bovine type III collagen and the collagen disclosed in CN114671946B have not shown obvious changes compared to 0 hours, while the collagen of the present invention has shown good migration, and the cells are almost 100% confluent at about 24 hours.

[0036] 3. The recombinant human type III collagen fermentation broth of the present invention can obtain recombinant human type III collagen with high purity through cation chromatography, EK enzyme digestion and anion chromatography steps. The SDS-PAGE results show that there are almost no impurity protein bands. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the local structure of the recombinant pET-32a-Kan vector

[0038] Figure 2 Colony selection chart

[0039] Figure 3 SDS-PAGE electrophoresis of induced expression fusion protein, where M: 10-245 kDa marker; Yin: 7-1 negative control before induction

[0040] Figure 4 Soluble SDS-PAGE electrophoresis, where M: 10-245 kDa marker; 5: supernatant sample after lysis; 6: precipitate sample after lysis

[0041] Figure 5 SDS-PAGE results, lane 1: Maker; lane 2: sample after cation chromatography; lane 3: sample after enzyme digestion; lane 4: sample after anion chromatography

[0042] Figure 6 Cell proliferation activity

[0043] Figure 7 Promoting cell migration ability DETAILED DESCRIPTION

[0044] 1. Definition:

[0045] BJ cells: male foreskin fibroblasts

[0046] IPTG: Isopropyl-β-D-thiogalactopyranoside

[0047] Kan: Kanamycin

[0048] LB medium: 10g neutral soy peptone, 10g sodium chloride and 5g yeast powder, dilute to 1000mL with purified water;

[0049] CCK-8: Cell Counting Kit-8

[0050] DMEM: Dulbecco's Modified Eagle Medium

[0051] 50% glycerol: The volume percentage of glycerol in glycerol aqueous solution is 50%;

[0052] EK enzyme: Enterokinase (EK)

[0053] TrxA: Thioredoxin

[0054] Carbon source: 50% (w / v) glucose in water

[0055] Nitrogen source: yeast extract powder (120.00±1.20 g) and neutral soy peptone (40.00±0.40 g), dilute to 500 ml with purified water;

[0056] Trace element solution (1L): contains 2.80g ferrous sulfate heptahydrate, 1.71g manganese sulfate monohydrate, 2.37g cobalt chloride hexahydrate, 1.13g calcium chloride dihydrate, 0.29g copper sulfate pentahydrate, 0.29g zinc sulfate heptahydrate, 1ml hydrochloric acid, and the rest is water;

[0057] Fermentation basal medium: 3.6 L of culture medium, including 44.85 g of yeast extract, 76.05 g of neutral soy peptone, 15.60 g of ammonium sulfate, 4.68 g of magnesium sulfate heptahydrate, 70.20 g of dipotassium hydrogen sulfate, 11.70 g of potassium dihydrogen phosphate, 400 μl of polypropylene glycol 2000, and the rest is water.

[0058] The concentration of the Tris-HCl solution involved in the present invention is expressed as the concentration of Tris, such as 50 mM Tris-HCl solution means that the concentration of Tris is 50 mM.

[0059] The Tris-HCl solution referred to in the present invention refers to a pH buffer system formed by adding HCl to Tris.

[0060] In the present invention, "+" means "and", such as: "50 mM Tris-HCl+10 mM imidazole+500 mM NaCl" means that the concentration of Tris-HCl in the solution is 50 mM, the concentration of imidazole is 10 mM, and the concentration of NaCl is 500 mM.

[0061] The recombinant pET32a(+)-Kan expression vector containing the recombinant human type III collagen target gene of the present invention is constructed from Nanjing GenScript Biotechnology Co., Ltd.

[0062] The protein obtained after induced expression or fermentation (i.e., before the EK enzyme cleavage step) in the present invention is a fusion protein, which is mainly composed of the recombinant human type III collagen (i.e., the target protein) of the present invention and thioredoxin, and the thioredoxin is derived from the pET-32a-Kan expression vector.

[0063] In the present invention, the fusion protein obtained after induction expression or fermentation is cut off by EK enzyme to obtain the target protein recombinant human type III collagen.

[0064] The amino acid sequence of the recombinant human type III collagen in the present invention is SEQ ID NO: 1:

[0065] GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD;

[0066] The recombinant human type III collagen described in the present invention is encoded by the gene sequence shown in SEQ ID NO: 2, wherein the sequence of SEQ ID NO: 2 is:

[0067]

[0068] 2. Source of reagents

[0069]

[0070] The reagents not specifically explained in the present invention are all known products and can be obtained by purchasing commercial products.

[0071] In order to describe the present invention more clearly and completely, the present invention is described below in conjunction with specific embodiments. The embodiments are only used to explain the present invention but not to limit the protection scope of the present invention.

[0072] Example 1 Design and expression of recombinant human type III collagen molecule

[0073] 1. Protein sequence design

[0074] Extract specific amino acid fragments of full-length human type III collagen

[0075] GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD,

[0076] Repeat 4 times to obtain sequence SEQ.ID.NO.1.

[0077] 2. Construction of recombinant expression vector

[0078] The coding nucleic acid sequence was reversely designed and codon optimized to obtain the DNA sequence SEQ ID NO: 2 encoding recombinant human type III collagen; the sequence 5' - CACCACCACCATCACCATGATGATGATGACAAG-3' was connected to the 5' end of the SEQ ID NO: 2 sequence, and then inserted into the restriction sites EcoR I and Kpn I of the expression vector pET-32a-Kan to obtain a recombinant expression vector containing a recombinant human type III collagen encoding gene, as shown in the structure attached Figure 1 shown.

[0079] 3. Construction and screening of expression strains

[0080] 1) Conversion

[0081] Take out the E. coli BL21 (DE3) competent cells stored at -80℃ and place them on ice. In the clean bench, add 0.5μl of the recombinant expression vector (concentration of 100 ng / μl) to 50μl of the competent cell suspension. Incubate on ice for 30 min. Heat shock at 42℃ for 90 s and then quickly incubate on ice for 5 min. Add 1 ml of antibiotic-free LB liquid culture medium and culture on a constant temperature shaking incubator at 37℃, 200 rpm for 60 min.

[0082] 2) Screening

[0083] The transformed bacteria obtained in step (2) were spread on LB resistance plates and inverted in a constant temperature incubator at 37°C for overnight culture. The next day, single colonies with rounded morphology and good growth status were randomly picked (such as those attached Figure 2 As shown in the figure, the cells were inoculated into 5 ml of LB resistance medium containing 50 μg / ml of Kan; the cells were cultured at 37 °C and 220 rpm for 4 h. Then, the cells were maintained in 1 ml / tube of glycerol at a final concentration of 25% (50% glycerol: bacterial liquid volume = 1:1) and stored in a -80 °C refrigerator.

[0084] 4. Inducible expression

[0085] Three groups of glycerol bacteria obtained in the recovery step 3, numbered 7-1, 7-2 and 7-3, were induced to express for 4 h in a shake flask at 37°C and 1.0 mM IPTG, and the induced bacterial liquid was taken for SDS-PAGE detection.

[0086] SDS-PAGE detection: Take 1 ml of the induced bacterial solution and centrifuge at 10,000 rpm for 5 min to collect the bacterial precipitate, add 1 ml of water to resuspend, take 40 μl of the suspension, add 10 μl of 5× Loading Buffer and mix well, boil in a 100℃ metal bath for 5 min, cool and centrifuge at 10,000 rpm for 5 min to obtain the supernatant. Add 5 μl marker and 20 μl sample supernatant to the comb hole respectively, keep constant voltage at 140 V for 60 min, stop running when bromophenol blue migrates to the bottom of the gel; take out the gel and use eStain L1 protein stainer for staining and decolorization, scan it with GS-900 calibration optical density instrument, and use software to analyze and calculate the integral percentage of sample band optical density.

[0087] As attached Figure 3 As shown, a fusion protein was expressed around 75 kDa, and the molecular weight was consistent with theoretical expectations.

[0088] Example 2 Analysis of water solubility of recombinant human type III collagen

[0089] Take 5 ml of the bacterial solution collected after induction expression in Example 1, centrifuge at 10,000 rpm for 5 min, collect the bacterial precipitate, resuspend with 5 ml of purified water, disrupt with an ultrasonic cell disruptor (550 W, ultrasound for 20 min) under ice bath, centrifuge at 10,000 rpm for 5 min, separate the bacterial precipitate and the supernatant. Perform SDS-PAGE detection respectively, wherein the SDS-PAGE detection process refers to that shown in Example 1.

[0090] As attached Figure 4 As shown, bacterial sediment samples (attached Figure 4 There is almost no fusion protein band in the supernatant sample (marked 6 in the attached Figure 4 Marked in 5) shows an obvious fusion protein band, indicating that the recombinant human type III collagen prepared by the present invention has excellent water solubility.

[0091] Example 3 Fermentation and purification of recombinant human type III collagen

[0092] 1. Fermentation process

[0093] 1 ml of the frozen glycerol bacteria of Example 1 was inoculated into 50 ml of LB medium containing 50 μg / ml Kan, and cultured at 37°C, 220 rpm for about 4 h. 3.6 ml of the seed solution was transferred into 360 ml of LB medium containing 50 μg / ml Kan, and cultured at 37°C, 220 rpm for about 4 h.

[0094] Add 40 ml of 50% glucose solution to a fermenter containing 3.6 L of basal medium, stir evenly, add 8 ml of trace element solution and 360 ml of seed solution, the initial culture volume is about 3.9 L, set the temperature to 37.0±1.0 ℃, pH to 7.0±0.2, and control the dissolved oxygen to be not less than 20%. After culturing for 2 hours, add carbon source and nitrogen source; after culturing for 5 hours, add IPTG solution to a final concentration of 1 mmol / L to start induction culture, 37.0±1.0℃, pH to 7.0±0.2, dissolved oxygen not less than 50%, induce for 4 hours, and obtain 5g / L of fusion protein.

[0095] 2. Purification process

[0096] 1) Cell disruption by centrifugation

[0097] Fermentation broth, according to the cell weight and lysis buffer (50 mM Tris-HCl + 10 mM imidazole + 500 mM NaCl, pH 8.0) ratio of about 1: 15 (W / V), add lysis buffer (50 mM Tris-HCl + 10 mM imidazole + 500 mM NaCl, pH 8.0) to the cells, and use a constant temperature magnetic stirrer with a rotor to fully resuspend the cell solution at 300r / min. After resuspension, filter with a 60-mesh sieve, and then lyse the cells: pressure 800bar-900bar, 2 times. The lyse solution is centrifuged at 6520r / min, 4 ° C, single centrifugation for 10min, centrifugation times 2 times, collect the supernatant, filter, and obtain the crude fusion protein.

[0098] 2) Cationic chromatography

[0099] Cationic chromatography: SP Sepharose Fast Flow was loaded into the chromatography column, regenerated with 50mM Tris-HCl +0.5M NaCl, pH 8.5±0.1, balanced with 50mM Tris-HCl pH8.5±0.1, loaded with the crude fusion protein in step 1), re-equilibrated with equilibration solution (50mM Tris-HCl, pH8.5±0.1) until the UV baseline was stable, and eluted with a high concentration salt solution 50mM Tris-HCl +0.5M NaCl to obtain the fusion protein.

[0100] 3) Enzyme digestion

[0101] The fusion protein was diluted with purified water to about 1 mg / ml and digested with EK enzyme at 25°C±2°C for 4 h, wherein the concentration of EK enzyme:protein = 2 IU:1 mg.

[0102] 4) Anion chromatography

[0103] UniGel-80Q was loaded into a chromatography column, regenerated with 50 mM Tris-HCl +0.5 M NaCl, pH 8.0±0.1, equilibrated with 50 mM Tris-HCl pH8.0±0.2 buffer, loaded with the digested sample, re-equilibrated with 50 mM Tris-HCl pH8.0±0.2 equilibration solution until the UV baseline was stable, eluted with 50 mM Tris-HCl +0.5 M NaCl, and the target protein was obtained for SDS-PAGE detection. The SDS-PAGE detection process was shown in Example 1.

[0104] As attached Figure 5 As shown, the purified target protein of the present invention substantially does not contain other impurity protein bands.

[0105] Example 4 Purification of recombinant human type III collagen

[0106] 1. Fermentation process

[0107] The crude fusion protein containing recombinant human type III collagen was obtained by referring to the fermentation process in Example 3.

[0108] 2. Purification process

[0109] 1) Cell disruption by centrifugation: refer to Example 3.

[0110] 2) Ni (nickel) affinity chromatography

[0111] Load NiBestarose 6FF into a chromatography column, equilibrate the column with 50 mM Tris-HCl + 0.15 M NaCl pH 8.0 ± 0.2 buffer, load the crude fusion protein in step 1), re-equilibrate with equilibration solution until the UV baseline is stable, and elute with 50 mM Tris-HCl + 250 mM imidazole to obtain the fusion protein.

[0112] 3) Enzyme digestion: refer to Example 3.

[0113] 4) Anion chromatography: refer to Example 3

[0114] The harvested anion chromatographic solution was subjected to SDS-PAGE detection as shown in Example 1, and this purification method also contained almost no foreign protein bands.

[0115] Example 5 Detection of biological activity of recombinant human type III collagen

[0116] Preparation of collagen solution:

[0117] (1) Preparation of bovine type III collagen solution:

[0118] Take bovine type III collagen, add PBS solution, stir electrically for 3 hours at a stirring speed of 100 rpm to obtain 5 mg / ml bovine type III collagen stock solution, filter sterilize, and freeze at -20°C. Dilute with DMEM medium to 4 concentrations of 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml and 0.5 mg / ml before the experiment, and perform corresponding tests.

[0119] (2) The present invention and CN114671946B recombinant human type III collagen preparation:

[0120] The target protein after anion chromatography in Example 3 of the present invention was diluted with DMEM medium to 4 concentrations of 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml and 0.5 mg / ml, and corresponding tests were performed.

[0121] The recombinant collagen prepared according to the method disclosed in Example 114671946B was diluted with DMEM culture medium to 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml and 0.5 mg / ml, and the corresponding tests were performed.

[0122] 1. Detection of cell proliferation activity:

[0123] The CCK-8 method was used to detect the effect of the collagen prepared in the present invention on the proliferation of BJ cells. The specific operation steps are as follows:

[0124] 1) Cell culture: BJ cells were cultured at 5 × 10 3 The cells were seeded in a 96-well plate (DMEM medium containing 10% FBS) at a density of 10 cells / well and cultured for 24 hours. The culture medium was discarded and the cells were washed 3 times with PBS.

[0125] 2) Cell treatment: 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml and 0.5 mg / ml of the recombinant human type III collagen solution of the present invention were prepared respectively, and 100 μL of collagen of different concentrations was added to the cells respectively; the same volume of DMEM medium was added to the control well (i.e., the collagen concentration was 0). After incubation for 24 hours at 37°C and 5% CO2, the supernatant was discarded, and 100 μL of 10% CCK-8 solution (serum-free DMEM) was added to each well, and then placed in a cell culture incubator for incubation for 2 hours.

[0126] 3) Detection: After taking out, shake lightly, draw 100 μL of the detection solution into a new ELISA plate, and measure the absorbance (OD) at 450 nm using a multifunctional ELISA reader. The absorbance value of the collagen group is At, the absorbance value of the control group is Ac, and the absorbance of the blank group is A0. Cell survival rate = (At-A0) / (Ac-A0)×100%.

[0127] In addition, bovine type III collagen and CN114671946B recombinant collagen were selected as the control group, and the cell culture, cell treatment and detection processes thereof were as described above.

[0128] As attached Figure 6As shown, it can be seen that the recombinant human type III collagen prepared by the present invention has an obvious effect of promoting cell proliferation, and its proliferation-promoting activity is about twice that of bovine type III collagen and CN114671946B collagen.

[0129] 2. Detection of cell migration:

[0130] The effect of recombinant collagen on BJ cell migration was observed under an inverted microscope. The specific steps are as follows:

[0131] 1) Cell culture and labeling: BJ cells were cultured at 5 × 10 5 The cells were seeded at a density of 100 cells in a 6-well plate (DMEM medium containing 10% FBS). After culturing for 24 hours, the medium was replaced with 2 ml of DMEM medium containing 1% FBS, 0.5 mg / ml bovine type III collagen, 0.5 mg / ml collagen prepared according to the method disclosed in Example 1 of CN114671946B, and 0.5 mg / ml collagen of the present invention, respectively.

[0132] 2) Scratch: Use a sterile 100 µL pipette tip to create a scratch of uniform width on the cell monolayer and continue to culture at 37°C and 5% CO2.

[0133] 3) Observation and recording: The well plates were removed after 0 h, 6 h, and 24 h, respectively, and washed three times with PBS to remove detached cells. The scratched areas were observed and photographed under an inverted microscope.

[0134] As attached Figure 7 As shown, under the same culture conditions, after about 6 hours, the cell migration abilities of the blank group (i.e., 1% FBS group, represented by NC), bovine type III collagen, and CN114671946B collagen did not show obvious differences, while the recombinant human type III collagen prepared by the present invention showed better migration; at the same time, the scratch gaps of the collagen of the present invention were almost 100% confluent after about 24 hours.

Claims

1. A recombinant human type III collagen, characterized in that: The amino acid sequence of type III collagen is: (GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEK GETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD) n The n is 4.

2. A nucleic acid molecule encoding the recombinant human type III collagen as claimed in claim 1, characterized in that: The DNA sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to claim 2.

4. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria comprises the recombinant expression vector as claimed in claim 3.

5. A method for preparing recombinant human type III collagen as claimed in claim 1, comprising the following steps: (1) Protein sequence design Extract specific amino acid fragments of full-length human type III collagen GAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKG ETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSD, Repeat 4 times to obtain the amino acid sequence of recombinant human type III collagen as shown in SEQ ID NO: 1; (2) Construction of recombinant expression vector Connect the sequence 5'-CACCACCACCATCACCATGATGATGATGACAAG-3' to the 5' end of the sequence of SEQ ID NO: 2, and then insert it into the restriction sites EcoR I and Kpn I of the expression vector pET-32a-Kan to obtain a recombinant expression vector containing a recombinant human type III collagen encoding gene; (3) Construction and screening of expression strains The recombinant expression vector described in step (2) is transferred into the host cell by heat shock method, and after plating overnight, the recombinant expression vector is selected and inoculated into LB resistance medium containing Kan to obtain the expression strain; (4) Induced expression and purification The expression strain obtained in step (3) is induced to express, the bacterial solution is collected, and purified to obtain recombinant human type III collagen. The preparation method according to claim 5 , wherein the host cell is Escherichia coli BL21 (DE3).

7. The preparation method according to claim 5, wherein the induction expression condition is 37°C, 1.0 mM IPTG induction for 4 hours.

8. The preparation method according to claim 5, wherein the purification condition is to purify the bacterial solution collected by induced expression according to the following steps in order: resuspending and disrupting the collected bacterial solution, collecting the supernatant by centrifugation, filtering, collecting the filtrate and then performing Ni affinity chromatography or cation chromatography, EK enzyme digestion, and finally anion chromatography to obtain recombinant human type III collagen.

9. Use of the recombinant human type III collagen as described in claim 1 in the preparation of medical device products; the medical device products are selected from: skin wound dressings, scar repair dressings, oral mucosa repair dressings, oral care dressings, vaginal repair dressings, vaginal care dressings, uterus repair dressings, uterus care dressings, nasal mucosa repair dressings, nasal care dressings and cavity lubrication dressings. One or more.

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