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

Abstract

The invention belongs to the field of biological engineering and biological materials, and provides a recombinant human III-type collagen which comprises an N-terminal sequence and a C-terminal sequence, the N-terminal sequence comprises a repetitive unit, and the amino acid sequence composition of the repetitive unit is shown as SEQ ID NO: 1 in a sequence table; the amino acid sequence composition of the C-terminal sequence is as shown in SEQ ID NO: 2 in the sequence table. The recombinant human III-type collagen provided by the invention basically does not contain endotoxin after being purified, and can be self-assembled into collagenous fibers, and the activity of the collagen is equivalent to that of naturally extracted animal collagen.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and biomaterials, and particularly relates to a recombinant human type III collagen, its preparation method and application. Background Technology

[0002] Collagen comprises 95% of dermal tissue. Its unique triple helix structure forms the basis of its physicochemical properties and biological activity, giving it high tensile strength, biodegradability, low antigenic activity, low irritation, low cytotoxicity, and the ability to promote cell growth, cell adhesion, and synergistic wound repair with newly formed cells and tissues when used as a framework for artificial organs or in wound dressings. Collagen's excellent physicochemical properties and biological activity make it a widely applicable biomedical material, currently used in dressings, regenerative medicine for artificial organs, tissue engineering, and bio-skincare.

[0003] Collagen preparation methods can be divided into animal-derived extraction and gene recombination engineering. Gene recombination engineering can overcome the pain points of animal-derived extraction in terms of production cost and safety, and is the mainstream of future technological development. Currently, recombinant collagen is mainly expressed using *E. coli* and yeast cells; however, the triple helix level of the expressed recombinant collagen is low, affecting its ability to perform its intended functions. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides a recombinant human type III collagen, its preparation method, and its applications.

[0005] This invention provides a recombinant human type III collagen, comprising an N-terminal sequence, wherein the N-terminal sequence comprises a repeating unit, the amino acid sequence of which is as shown in SEQ ID NO:1 in the sequence listing; or: the amino acid sequence of which is obtained by mutating, inserting, or deleting one or more amino acid residues from the sequence shown in SEQ ID NO:1 in the sequence listing; or: the amino acid sequence of which has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:1 in the sequence listing; the collagen further comprises a C-terminal sequence, the amino acid sequence of which is as shown in SEQ ID NO:2 in the sequence listing; or: the C-terminal sequence is obtained by mutating, inserting, or deleting one or more amino acid residues from SEQ ID NO:2 in the sequence listing.

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] 1. This invention uses CHO cells as host cells to express recombinant human type III collagen, and the purified collagen is essentially free of endotoxins.

[0008] 2. The recombinant humanized type III collagen provided by this invention has a distinct triple helix structure and can self-assemble into collagen fibers.

[0009] 3. The recombinant humanized type III collagen provided by this invention has excellent biological activity, and experimental data show that its activity is comparable to that of naturally extracted animal collagen.

[0010] 4. The preparation method of the present invention is simple to operate and saves resources; the various operation steps of the identification method work together to achieve excellent results. Attached Figure Description

[0011] Figure 1 This is an SDS-PAGE electrophoresis image of recombinant human type III collagen ON1-1, ON1-2 and ON1-4 from Example 1.

[0012] Figure 2 The chromatograms of recombinant human type III collagen ON1-1, ON1-2 and ON1-4 were obtained for detection in Example 1 using circular dichroism chromatography.

[0013] Figure 3 Transmission electron microscopy images of recombinant human type III collagen ON1-1, ON1-2 and ON1-4 for detection in Example 1.

[0014] Figure 4 This is a bar chart showing the bioactivity results of recombinant human type III collagen ON1-1, ON1-2, and ON1-4 in Example 1. The left half of the chart shows the cell adhesion activity results, and the right half shows the cell proliferation activity results. Detailed Implementation

[0015] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0016] In a first aspect, the present invention provides a recombinant human type III collagen, the collagen comprising an N-terminal sequence, the N-terminal sequence comprising repeating units, the amino acid sequence composition of the repeating units being shown in SEQ ID NO:1 of the sequence listing:

[0017] The amino acid sequence of the repeating unit can also be the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:1 in the sequence listing.

[0018] The amino acid sequence of the repeating unit may also have greater than or equal to 95% homology with the sequence shown in SEQ ID NO:1 in the sequence listing.

[0019] The number of repeating units is 1 to 8.

[0020] The repeating units, which are more than one, are directly connected in sequence or separated by one or more amino acid residues.

[0021] The collagen also includes a C-terminal sequence. The amino acid sequence of the C-terminal sequence is GAPGPCCGG, as shown in SEQ ID NO:2 in the sequence listing.

[0022] The C-terminal sequence can also be the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues from SEQ ID NO:2 in the sequence listing.

[0023] The C-terminal sequence can be directly attached to the C-terminus of the last repeating unit of the N-terminal sequence, or it can be separated from the C-terminus of the last repeating unit of the N-terminal sequence by one or more amino acid residues.

[0024] In one specific embodiment, the N-terminal sequence of the collagen (ON1-1) includes one repeating unit, the C-terminus of which is connected to the C-terminal sequence, and the amino acid sequence composition of the collagen (ON1-1) is as shown in SEQ ID NO:3 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:3 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:3 in the sequence listing.

[0025] In another specific embodiment, the N-terminal sequence of the collagen (ON1-2) includes two repeating units, and the C-terminus of the second repeating unit is connected to the C-terminal sequence. The amino acid sequence composition of the collagen (ON1-2) is as shown in SEQ ID NO:4 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:4 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:4 in the sequence listing.

[0026] In another specific embodiment, the N-terminal sequence of the collagen (ON1-4) includes four repeating units, and the C-terminus of the fourth repeating unit is connected to the C-terminal sequence. The amino acid sequence composition of the collagen (ON1-4) is as shown in SEQ ID NO:5 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:5 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:5 in the sequence listing.

[0027] In a second aspect, the present invention provides a polynucleotide sequence encoding the recombinant human type III collagen described in the first aspect above.

[0028] According to a second aspect of the present invention, the polynucleotide sequence encoding the recombinant human type III collagen comprises a polynucleotide sequence encoding the repeating sequence of the N-terminal sequence of the recombinant human type III collagen, and a polynucleotide sequence encoding the C-terminal sequence of the recombinant human type III collagen; wherein:

[0029] The polynucleotide sequence composition of the repeating sequence encoding the N-terminal sequence of the recombinant human type III collagen is shown in SEQ ID NO:6 in the sequence listing;

[0030] Alternatively: the polynucleotide sequence of the repeating sequence encoding the N-terminal sequence of the recombinant human type III collagen constitutes a sequence having greater than or equal to 95% homology with the sequence shown in SEQ ID NO:6 in the sequence listing;

[0031] Alternatively: The polynucleotide sequence of the repeating sequence encoding the N-terminal sequence of the recombinant human type III collagen is composed of a nucleotide sequence encoding a protein having the same function as recombinant human type III collagen (ON1-1) obtained by substitution, deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:6 in the sequence listing.

[0032] The polynucleotide sequence composition encoding the C-terminal sequence of the recombinant human type III collagen is shown in SEQ ID NO:7 in the sequence listing;

[0033] Alternatively: The multinucleotide sequence encoding the C-terminal sequence of the recombinant human type III collagen is composed of a sequence having greater than or equal to 95% homology with the sequence shown in SEQ ID NO:7 in the sequence listing;

[0034] Alternatively: The polynucleotide sequence encoding the C-terminal sequence of the recombinant human type III collagen is composed of a nucleotide sequence encoding a protein having the same function as the C-terminal sequence of recombinant human type III collagen, obtained by substitution, deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:7 in the sequence listing.

[0035] In one specific embodiment, the polynucleotide sequence encoding the recombinant human type III collagen (ON1-1) is shown in SEQ ID NO:8 in the sequence listing.

[0036] Alternatively: the polynucleotide sequence encoding the recombinant human type III collagen (ON1-1) has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:8 in the sequence listing;

[0037] Alternatively: The polynucleotide sequence encoding the recombinant human type III collagen (ON1-1) is obtained by substituting, deleting, and / or adding one or more nucleotides based on the sequence shown in SEQ ID NO:8 in the sequence listing, and encoding a protein having the same function as recombinant human type III collagen (ON1-1).

[0038] In another specific embodiment, the polynucleotide sequence encoding the recombinant human type III collagen (ON1-2) is shown in SEQ ID NO:9 in the sequence listing;

[0039] Alternatively: the polynucleotide sequence encoding the recombinant human type III collagen (ON1-2) has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:9 in the sequence listing;

[0040] Alternatively: The polynucleotide sequence encoding the recombinant human type III collagen (ON1-2) is obtained by substituting, deleting, and / or adding one or more nucleotides based on the sequence shown in SEQ ID NO:9 in the sequence listing, and encoding a protein having the same function as recombinant human type III collagen (ON1-2).

[0041] In another specific embodiment, the polynucleotide sequence encoding the recombinant human type III collagen (ON1-4) is shown in SEQ ID NO:10 in the sequence listing;

[0042] Alternatively: the polynucleotide sequence encoding the recombinant human type III collagen (ON1-4) has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:10 in the sequence listing;

[0043] Alternatively: The polynucleotide sequence encoding the recombinant human type III collagen (ON1-4) is obtained by substituting, deleting, and / or adding one or more nucleotides based on the sequence shown in SEQ ID NO:10 in the sequence listing, and encoding a protein having the same function as recombinant human type III collagen (ON1-4).

[0044] In a third aspect, the present invention provides an expression vector containing the polynucleotides described in the second aspect above. The framework vector of the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, for example, a pEE12.4 plasmid vector, a pXC17.4 plasmid vector, or other expression vectors.

[0045] In a fourth aspect, the present invention provides a host cell containing the expression vector described in the third aspect above. The host cell includes: mammalian cells, human skin cells, yeast, or *Escherichia coli*. In a preferred embodiment, the mammalian cell is a CHO cell (e.g., CHO-K1 cell, CHO-S cell, CHO-DG44 cell) or a HEK293 cell; the human skin cell is preferably an HSF cell; and the yeast is preferably *Pichia pastoris* or *Saccharomyces cerevisiae*.

[0046] In a fifth aspect, the present invention provides a method for preparing recombinant human type III collagen, the method comprising expressing, in expression cells, a constructed expression vector bearing nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 respectively, to obtain recombinant human type III collagen ON1-1, ON1-2, and ON1-4 respectively.

[0047] According to a fifth aspect of the present invention, the method may include the following steps:

[0048] Vector construction steps: Insert the above nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 into the above framework vector to obtain expression vectors with nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 respectively.

[0049] Transformation and expression steps: The above expression vectors were transformed into expression cells, and then expressed separately to obtain transformed cells containing recombinant human type III collagen ON1-1, ON1-2 and ON1-4.

[0050] The expression vectors mentioned above can be pEE12.4 plasmid vector, pXC17.4 plasmid vector, or other expression vectors.

[0051] The aforementioned expressing cells can be the host cells described in the fourth aspect above.

[0052] Purification steps for the target protein: The transformed cells containing recombinant human type III collagen ON1-1, ON1-2 and ON1-4 were subjected to protein purification treatment to obtain recombinant human type III collagen ON1-1, ON1-2 and ON1-4.

[0053] Preferably, the above protein purification process includes: centrifugation, concentration, filtration, and chromatography.

[0054] The above-mentioned concentration process preferably uses a tangential flow system.

[0055] The above filtration process preferably uses a 0.22 μm ultrafiltration membrane.

[0056] The above-mentioned chromatographic treatment preferably uses a cation chromatography column; the above-mentioned chromatographic treatment preferably includes: first equilibrating the cation chromatography column, then loading the sample, then equilibrating again, and then gradient linear elution.

[0057] In a sixth aspect, the present invention provides a composition comprising the recombinant human type III collagen described in the first aspect above. The composition can be used to manufacture medical materials, medical devices, and cosmetic medical materials.

[0058] In a seventh aspect, the present invention provides a gel comprising the recombinant human type III collagen described in the first aspect above, and / or the composition described in the sixth aspect above. The gel can be used to manufacture medical materials, medical devices, and medical aesthetic materials.

[0059] In an eighth aspect, the present invention provides the application of the recombinant human type III collagen described in the first aspect above, and / or the polynucleotide sequence described in the second aspect above, and / or the expression vector described in the third aspect above, and / or the host cell described in the fourth aspect above, and / or the preparation method described in the fifth aspect above, and / or the composition described in the sixth aspect above, and / or the gel described in the seventh aspect above, in the preparation of medical materials, medical devices, and medical aesthetic materials.

[0060] Example 1

[0061] This embodiment describes the preparation of recombinant human type III collagen.

[0062] S1. Nucleotide Sequence Synthesis: The amino acid sequences SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 of collagen ON1-1, ON1-2, and ON1-4, respectively, were codon-optimized according to the codon preference of CHO cells to obtain the corresponding nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Then, adapters for the restriction enzymes Hind III and EcoRI were added to both ends of the above nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, to synthesize the adapter-linked fragments SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0063] The amino acid sequence of collagen ON1-1 corresponds to SEQ ID NO:3 and the nucleotide sequence of collagen ON1-2 corresponds to SEQ ID NO:4 and the nucleotide sequence of collagen ON1-4 corresponds to SEQ ID NO:5 and the nucleotide sequence of collagen ON1-4 corresponds to SEQ ID NO:10.

[0064] S2. Expression vector construction: Using plasmid vector pEE12.4 (purchased from Shanghai Zeye Biotechnology Co., Ltd.) as the framework vector, the plasmid vector pEE12.4 was double-digested with restriction enzymes Hind III and EcoRI to obtain a large fragment; then, the above-mentioned fragments with adapters SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 were respectively ligated with the above-mentioned large fragment to obtain expression vectors ON1-1-pEE12.4, ON1-2-pEE12.4, and ON1-4-pEE12.4.

[0065] S3, Protein Expression:

[0066] (1) Resuscitate CHO-K1 cells in advance with CHO basic culture medium; then passage the resuscitated CHO-K1 cells for 3 generations. When the cell viability reaches 98%, they can be used for electroporation and kept for later use.

[0067] The CHO-K1 cell line described above was obtained from the European Standard Cell Collection Center (ECACC). The basic culture of the CHO cells was performed using CD CHO medium (catalog number: 12490025) purchased from Gibco.

[0068] (2) The constructed expression vectors ON1-1-pEE12.4, ON1-2-pEE12.4 and ON1-4-pEE12.4 were electroporated into CHO-K1 cells using an electroporator.

[0069] The aforementioned electrostatic precipitator is a GenePulser Xcell, brand name. Its parameters include: voltage 300V, capacitance 900μF, and resistance ∞.

[0070] (3) After electroporation for 24 hours, the CHO-K1 cells were replaced with pressurized culture medium.

[0071] The pressurized culture medium described above was prepared by adding L-methionine sulfoxide (MSX) to a final concentration of 50 μM, based on the basic CHO culture. MSX was purchased from Sigma, catalog number M5379.

[0072] (4) When the cell viability reaches 95% or more, CHO-K1 cells are placed in expression medium and cultured for 10 days to obtain CHO transformed cells expressing recombinant human type III collagen ON1-1, ON1-2 and ON1-4.

[0073] The expression medium used was Dynamis medium (catalog number: A2617501) purchased from Gibco. The expression culture conditions were: temperature 37℃, humidity 70%, CO2 concentration 8%, and rotation speed 120 rpm.

[0074] S4. Protein purification:

[0075] (1) Harvest the CHO-transformed cells and centrifuge them at 15000g for 30 minutes in a high-speed centrifuge to harvest the cell supernatant (i.e., supernatant liquid).

[0076] (2) The harvested cell supernatant was concentrated using tangential flow and adjusted to pH 6.0.

[0077] The tangential flow system described above uses Sartorius brand membranes; the ultrafiltration membranes described above were purchased from MERK, with a specification of 10kD.

[0078] (3) The concentrated cell supernatant was filtered through a 0.22 μm ultrafiltration membrane to obtain the filtrate.

[0079] (4) The above filtrate was subjected to cation chromatography to obtain an eluent containing the target protein.

[0080] The process includes: first, equilibrating the cation exchange column with 3 column volumes of equilibration buffer, then loading the filtrate (i.e., the processed protein) onto the column; then rinsing the column with 2 column volumes of equilibration buffer; and finally eluting the target protein with 10 column volumes of elution buffer in a linear gradient from 0% to 100%, to obtain the target proteins: recombinant human type III collagen ON1-1, ON1-2, and ON1-4.

[0081] The above-mentioned cation chromatography column uses Diamond MMC cation chromatography packing material.

[0082] The equilibration buffer was 50 mM Tris, pH 8.0. The elution buffer was 50 mM Tris, 1 M NaCl, pH 8.0.

[0083] (5) After elution, SDS-PAGE electrophoresis was used to detect the purity of each target protein in the elution buffer containing the target protein.

[0084] The theoretical molecular weight of ON1-1 is 25.7 kDa, that of ON1-2 is 50.5 kDa, and that of ON1-4 is 99.9 kDa. The purification results of recombinant human type III collagen ON1-1, ON1-2, and ON1-4 are as follows: Figure 1 As shown. Because CHO cells modified the target protein through glycosylation and other processes, the molecular weights were all higher than their theoretical values; for example... Figure 1 As shown, in the SDS electrophoresis image, ON1-1 shows a size between 40 and 50 kD, ON1-2 shows a size between 40 and 70 kD in the SDS gel, and ON1-4 shows a size between 140 and 200 kD in the SDS gel.

[0085] Detection Example 1

[0086] This test example describes the detection results of recombinant human type III collagen ON1-1, ON1-2, and ON1-4 prepared in Example 1.

[0087] I. Circular dichroism spectroscopy for detecting the triple helix structure of recombinant human type III collagen

[0088] Recombinant human type III collagen ON1-1, ON1-2, and ON1-4 were diluted to a concentration of 0.5 mg / mL with PBS, and their circular dichroism (CD) spectra were detected. Instrument parameters were set to room temperature (25℃), wavelength range of 190-260 nm, and measurement intervals of 1 nm. Results are as follows: Figure 2The main method for qualitative detection of the triple helix structure of collagen is circular dichroism chromatography, which is characterized by a maximum negative peak at approximately 198 nm and a maximum positive peak at approximately 220 nm. The ratio of the absolute values ​​of the maximum positive peak at 220 nm to the maximum negative peak at 198 nm reflects the degree of the triple helix structure of the collagen; the larger the ratio, the higher the proportion of the triple helix structure. Figure 2 ON1-1 did not exhibit this characteristic. ON1-2 showed a distinct triple helix feature with a ratio of 0.11; ON1-4 also exhibited this feature with a ratio of 0.045. The ratio of ON1-4 was much lower than that of ON1-2, indicating that it contained far less collagen with a triple helix structure than ON1-2. This demonstrates that ON1-2 has a distinct triple helix structure, and the triple helix of ON1-4 also exhibits a triple helix structure.

[0089] II. Transmission Electron Microscopy Characterization

[0090] Recombinant human type III collagen ON1-1, ON1-2, and ON1-4 were diluted with PBS to a concentration of 0.5 mg / mL for electron microscopy sample preparation. 100 μL of each of ON1-1, ON1-2, and ON1-4 was placed on parchment paper to form small droplets. A copper mesh was carefully picked up with tweezers and placed upside down on the liquid, allowing it to stand at room temperature for 5 min. After 5 min, the copper sheet was removed and placed on filter paper, then baked under a tungsten filament lamp for 20 min to completely evaporate the moisture. A drop of uranium acetate was added to the dried copper sheet, and it was then dried again under a tungsten filament lamp. The dried copper sheet was placed in a dust-free sample holder and fixed in place. The entire sample holder was then inserted into the transmission electron microscope for imaging. The results are shown below. Figure 3 As shown, the recombinant human type III collagen ON1-2 prepared by this invention can self-assemble into longer collagen fibers; the collagen fibers self-assembled by ON1-4 are shorter. Although no longer fibers were observed, it cannot be ruled out that ON1-4 can also self-assemble into longer collagen fibers; while ON1-1 does not show triple helix characteristics, so it cannot form higher-order structures, and no morphology with its higher-order structure was detected in electron microscopy.

[0091] III. Bioactivity Detection of Recombinant Human Type III Collagen

[0092] Experimental groups: PBS negative control group, 0.1 mg / mL ON1-2 and ON1-4 collagen protein experimental group, and bovine type I collagen positive control group.

[0093] 1. Cell adhesion activity assay

[0094] NIH-3T3 cell lines frozen in liquid nitrogen were retrieved and thawed in DMEM complete medium. When cell confluence reached 90%–95%, a cell suspension was prepared for use. In a 96-well plate, 100 μL of the corresponding sample was added to each well according to the grouping. The plate was incubated at 37°C with 5% CO2 for 4 h, and the liquid in the wells was aspirated, taking care not to touch the collagen coating at the bottom. Then, 100 μL of 1% BSA-PBS solution was added and the plate was blocked at 37°C with 5% CO2 for 1 h. The liquid in the wells was aspirated, and all coated wells were washed with PBS, repeated three times. 5 × 10⁵ cells were added to each well. 4 NIH / 3T3 cells in good culture condition were incubated at 37°C and 5% CO2 for 5 hours. The relative adhesion rate of the cells was detected using an LDH detection kit. Figure 4 As shown on the left side, the relative adhesion rates of each component are as follows: PBS is 7.75, ON1-4 is 68.50, ON1-2 is 93.88, and bovine type I collagen is 89.46. It can be seen that the relative adhesion rate of ON1-2 is greater than that of bovine type I collagen, and the relative adhesion rate of ON1-4 reaches about 70% of that of bovine type I collagen. Therefore, both ON1-2 and ON1-4 proteins have good adhesive activity.

[0095] 2. Cell proliferation activity detection

[0096] HSF cell lines frozen in liquid nitrogen were retrieved and thawed in DMEM complete medium. In a 96-well plate, 100 μL of the corresponding sample was added to each well according to the grouping. The plate was incubated at 37°C and 5% CO2 for 4 h, then the liquid in the wells was aspirated, taking care not to touch the collagen coating at the bottom. Then, 100 μL of 1% BSA-PBS solution was added and the plate was blocked at 37°C and 5% CO2 for 1 h. The liquid in the wells was aspirated, and all coated wells were washed with PBS, repeated three times. 1×10⁻⁶ cells were added to each well. 4 HSF cells in good culture condition were cultured at 37°C and 5% CO2 for 48 hours. 10 μL of CCK-8 solution was added to each well, and after incubation at 37°C for 1 hour, the absorbance at OD450 nm was measured to calculate the relative cell proliferation activity. Figure 4 As shown on the right, the relative proliferative activities of each protein are as follows: PBS 99.23, ON1-4 125.38, ON1-2 165.78, and bovine type I collagen 157.89. It can be seen that the relative proliferative activity of ON1-2 is greater than that of bovine type I collagen, and the relative adhesion rate of ON1-4 reaches about 75% of that of bovine type I collagen. Therefore, both ON1-2 and ON1-4 proteins have good relative proliferative activities.

[0097] In summary, the recombinant human type III collagen prepared by this invention has cell adhesion and proliferation activities that are basically equivalent to bovine type I collagen.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant human type III collagen, comprising an N-terminal sequence, characterized in that: The N-terminal sequence includes a repeating unit, the amino acid sequence of which is shown in SEQ ID NO:1 in the sequence listing; Alternatively: The amino acid sequence of the repeating unit is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:1 in the sequence listing; Alternatively: The amino acid sequence of the repeating unit has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:1 in the sequence listing.

2. The recombinant human type III collagen according to claim 1, characterized in that: The collagen also includes a C-terminal sequence; The amino acid sequence composition of the C-terminal sequence is shown in SEQ ID NO:2 in the sequence listing; Alternatively: The C-terminal sequence is the amino acid sequence obtained by mutating, inserting, or deleting one or more amino acid residues from SEQ ID NO:2 in the sequence listing; Preferably, the C-terminal sequence is directly connected to the C-terminal of the last repeating unit of the N-terminal sequence; Alternatively: There is one or more amino acids between the C-terminus of the last repeating unit of the C-terminal sequence and the N-terminal sequence; Preferably, the number of repeating units is 1 to 8; Preferably, more than one repeating unit is directly connected sequentially, or separated by one or more amino acid residues; Preferably, the N-terminal sequence of the collagen includes one repeating unit, and the C-terminus of the repeating unit is connected to the C-terminal sequence; the amino acid sequence composition of the collagen is as shown in SEQ ID NO:3 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:3 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:3 in the sequence listing. Preferably, the N-terminal sequence of the collagen includes two repeating units, and the C-terminus of each repeating unit is connected to the C-terminal sequence; the amino acid sequence composition of the collagen is as shown in SEQ ID NO:4 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting, or deleting one or more amino acid residues from the sequence shown in SEQ ID NO:4 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:4 in the sequence listing. Preferably, the N-terminal sequence of the collagen includes four repeating units, and the C-terminus of each repeating unit is connected to the C-terminal sequence; the amino acid sequence composition of the collagen is as shown in SEQ ID NO:5 in the sequence listing; or: the amino acid sequence composition of the collagen is the amino acid sequence obtained by mutating, inserting or deleting one or more amino acid residues of the sequence shown in SEQ ID NO:5 in the sequence listing; or: the amino acid sequence of the collagen has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:5 in the sequence listing.

3. A polynucleotide sequence encoding the recombinant human type III collagen as described in claim 1 or 2, characterized in that: The polynucleotide sequence encoding the recombinant human type III collagen includes a polynucleotide sequence encoding a repeating sequence of the recombinant human type III collagen and a polynucleotide sequence encoding the C-terminal sequence of the recombinant human type III collagen; wherein: The polynucleotide sequence of the repeating sequence encoding the N-terminal sequence of recombinant human type III collagen is as shown in SEQ ID NO:6 in the sequence listing; or: has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:6 in the sequence listing; or: is a nucleotide sequence encoding a protein having the same function as recombinant human type III collagen obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:6 in the sequence listing. The polynucleotide sequence encoding the C-terminal sequence of recombinant human type III collagen is as shown in SEQ ID NO:7 in the sequence listing; or: has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:7 in the sequence listing; or: is a nucleotide sequence encoding a protein having the same function as the C-terminal sequence of recombinant human type III collagen, obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:7 in the sequence listing.

4. The polynucleotide sequence encoding recombinant human type III collagen according to claim 3, characterized in that: The polynucleotide sequence encoding recombinant human type III collagen is shown in SEQ ID NO:8 in the sequence listing; or: has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:8 in the sequence listing; or: a nucleotide sequence encoding a protein with the same function as recombinant human type III collagen ON1-1, obtained by substitution, deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:8 in the sequence listing. Preferably, the polynucleotide sequence encoding recombinant human type III collagen is as shown in SEQ ID NO:9 in the sequence listing; or: has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:9 in the sequence listing; or: is a nucleotide sequence encoding a protein with the same function as recombinant human type III collagen ON1-2, obtained by substitution, deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:9 in the sequence listing. Preferably, the polynucleotide sequence encoding recombinant human type III collagen is as shown in SEQ ID NO:10 in the sequence listing; or: has greater than or equal to 95% homology with the sequence shown in SEQ ID NO:10 in the sequence listing; or: is a nucleotide sequence encoding a protein having the same function as recombinant human type III collagen ON1-4, obtained by substitution, deletion, and / or addition of one or more nucleotides based on the sequence shown in SEQ ID NO:10 in the sequence listing.

5. An expression vector containing a multinucleotide sequence encoding the recombinant human type III collagen as described in claim 3 or 4; Preferably, the expression vector is a pEE12.4 plasmid vector or a pXC17.4 plasmid vector.

6. A host cell containing an expression vector expressing the polynucleotide sequence encoding recombinant human type III collagen as described in claim 5, characterized in that: The host cells include: mammalian cells, human skin cells, yeast, or Escherichia coli; Preferably, the mammalian cells are CHO cells or HEK293 cells; the CHO cells are further preferably CHO-K1 cells, CHO-S cells, or CHO-DG44 cells. Preferably, the human skin cells are HSF cells; Preferably, the yeast is Pichia pastoris or Saccharomyces cerevisiae.

7. A method for preparing recombinant human type III collagen, the method comprising expressing the constructed expression vectors bearing nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 respectively in expression cells to obtain recombinant human type III collagen ON1-1, ON1-2, and ON1-4; Preferably, the method includes the following steps: Vector construction steps: Insert the nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 into the framework vector to obtain expression vectors containing the nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; Transformation and expression steps: The expression vectors containing nucleotide sequences SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 were transformed into expression cells, and then expressed and treated to obtain transformed cells containing recombinant human type III collagen ON1-1, ON1-2, and ON1-4. Purification steps for the target protein: The transformed cells containing recombinant human type III collagen ON1-1, ON1-2 and ON1-4 were subjected to protein purification treatment to obtain recombinant human type III collagen ON1-1, ON1-2 and ON1-4. Preferably, the purification process of the target protein includes: Centrifugation, concentration, filtration, and chromatography.

8. A composition comprising the recombinant human type III collagen as described in claim 1 or 2.

9. A gel comprising the recombinant human type III collagen of claim 1 or 2 and / or the composition of claim 8.

10. The use of the recombinant human type III collagen of claim 1 or 2, and / or the polynucleotide sequence of claim 3 or 4, and / or the expression vector of claim 5, and / or the host cell of claim 6, and / or the recombinant human type III collagen obtained by the preparation method of claim 7, and / or the composition of claim 8, and / or the gel of claim 9, in the preparation of medical materials, medical devices, and cosmetic materials.

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