Recombinant human VII type collagen as well as preparation method and application thereof

Abstract

According to the recombinant human VII type collagen as well as the preparation method and the application thereof, nucleotide optimized by codons is recombined into plasmids, and the plasmids are expressed in host cells, so that the recombinant human VII type collagen can be efficiently obtained; the recombinant human VII type collagen has collagen activity for promoting cell proliferation and cell adhesion, can inhibit metalloproteinase expression, has a cell repair effect, and can be used as a cell repair product; the method can be widely applied to food, cosmetics, health care products or medical equipment products.

Description

Technical Field

[0001] This application relates to the technical field of biotechnology and genetic engineering, and particularly relates to a recombinant human type VII collagen, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for about 30% of the total human protein; it is the main component of animal connective tissue, and it has now been proven that collagen hydrolysates have significant biological activities. There are relatively many types of collagen, and currently common types include type I, II, III, V, VII, XVII, etc. Among them, type VII collagen is mainly synthesized by keratinocytes and fibroblasts. Type VII collagen contains 2944 amino acids and consists of a central collagen domain and non-collagen nc-1 and nc-2 domains on both sides. The central collagen structure presents a typical collagen gly-x-y repeating structure sequence. Compared with other collagens, in the triple helix domain of type VII collagen, there is a "hinge" region composed of 39 amino acids, and this region is easily digested and hydrolyzed by pepsin. As a main component of the anchoring fibril structure, it can bind to type I collagen and type III collagen, providing stability for the interstitial membrane and basement membrane structures, and is crucial for maintaining the function and stability of the extracellular matrix.

[0003] However, the full-length molecular weight of type VII collagen is relatively large, and it is difficult to recombinantly express it in vitro. Currently, most researchers usually intercept partial fragments for recombinant expression, and may also add purification tags and residual secretion peptide signal sequences during the coding frame design, resulting in the final obtained collagen not being completely humanized collagen. Therefore, designing and developing a simple operation method for preparing recombinant type VII humanized collagen and its application has certain prospects and values. Summary of the Invention

[0004] The embodiments of this application provide a recombinant human type VII collagen, a preparation method thereof, and an application thereof to solve the problems existing in the related technologies. The technical solutions are as follows:

[0005] In the first aspect, the embodiments of this application provide a recombinant human type VII collagen, and the recombinant human type VII collagen includes:

[0006] a) an amino acid sequence as shown in SEQ ID No.1; or

[0007] b) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO.1, and it retains the collagen activity of the amino acid sequence of SEQ ID NO.1.

[0008] SEQ ID No.1:

[0009] GDKGDPGVGLPGPRGERGEPGIRGEDGRPGQEGPRGLTGPPGSRGERGEKGDVGSAGLKGDKGDSAVILGPPGPRGAKGDMGERGPRGLDGDKGPRGDNGDPGDKGSKGEPGDKGSAGLPGLRGLLGPQGQPGAAGIPGDPGSPGKDGVPGIRGEKGDVGFMGPRGLKGERGVKGACGLDGEKGDK。

[0010] In a second aspect, an embodiment of the present application provides a nucleotide encoding the above-mentioned recombinant type VII collagen, and the nucleotide is a nucleotide sequence optimized by codons; the nucleotide is as shown in SEQ ID No. 2;

[0011] SEQ ID No. 2:

[0012] atgggggataaaggagaccccggagtaggtctaccgggcccacgtggtgaacgcggcgaaccgggcatccgcggcgaagatggtcgtccgggccaagaaggtccgcgtggcttgaccggtcctccgggcagccgtggcgagcgcggtgagaagggtgacgttggtagcgctggactgaaaggtgacaagggcgacagcgctgtgattctgggcccaccgggtccgcgtggcgcgaaaggcgacatgggtgagcgcggtccgagaggcctggatggcgacaaaggtccgcgtggcgacaacggtgatccgggtgataagggctcgaaaggcgagccgggcgacaagggctctgcgggtctgccgggtctgcgtggcttgctgggtccacagggtcagccgggtgcggcaggcattccgggcgacccgggctccccgggtaaagatggtgttccggggatccgtggtgagaaaggtgacgtgggtttcatgggtccgcgtggtctcaagggtgagcgcggcgtcaaaggagcctgcggtttagatggtgaaaagggtgataagtaa。

[0013] The gene sequence of the recombinant type VII collagen of the present invention has been codon-optimized. After constructing a recombinant plasmid, an Escherichia coli expression system is used. The expressed recombinant type VII collagen is soluble, with an expression level as high as 30.32%. It has no protein purification tag and has no impact on the protein structure and function, and has the complete structure and function of recombinant type VII collagen.

[0014] In a third aspect, an embodiment of the present application provides a recombinant vector, and the recombinant vector is a recombinant plasmid, and the recombinant plasmid contains the above-mentioned nucleotides.

[0015] In one embodiment, the plasmid vector is C7-pET-30b(+).

[0016] In a fourth aspect, an embodiment of the present application provides a host cell with high expression, and the host cell with high expression contains the above-mentioned nucleotides; or the above-mentioned recombinant plasmid.

[0017] In one embodiment, the host cell with high expression is obtained by introducing the above-mentioned recombinant plasmid into a host.

[0018] In one embodiment, the host cell is Escherichia coli LTHINK(DE3) competent cells.

[0019] In a fifth aspect, an embodiment of the present application provides a method for preparing recombinant human type VII collagen. The above-mentioned host cell with high expression is used to express the recombinant type III collagen, and then separation and purification are carried out to obtain the recombinant human type VII collagen.

[0020] The present application realizes the high-yield expression of recombinant human type VII collagen by constructing and screening a host cell with high expression (genetic engineering strain). The expression level is 30%, which is the highest reported yield of recombinant human type VII collagen at present, and solves the technical problems of its low yield and high cost.

[0021] In one embodiment, the expression using the host cell refers to culturing the host cell, and the culture medium and culture conditions are well-known to those skilled in the art. For the expression mode, the present application does not make any restrictions, and it can be confirmed according to needs. For example, the expression mode is induced expression. The methods for separation and purification include salting-out method, ultrafiltration method, affinity chromatography method, gel filtration chromatography method, chromatographic chromatography method, acid-base precipitation method and membrane separation method. Preferably, the method for separation and purification is affinity chromatography method.

[0022] Sixth aspect, embodiments of the present application provide a cell repair reagent, which contains the recombinant human type VII collagen described above, or the recombinant human type VII collagen encoded by the nucleotide; or the recombinant human type VII collagen expressed by the highly expressed host cell.

[0023] In one embodiment, the concentration of the recombinant human type VII collagen in the cell repair reagent is 0.5 mg / mL - 1 mg / mL.

[0024] Seventh aspect, embodiments of the present application provide the application of the recombinant human type VII collagen described above, the recombinant human type VII collagen encoded by the nucleotide, and the recombinant human type VII collagen expressed by the highly expressed host cell in the preparation of cell repair reagents; their application in food, cosmetics, health products or medical device products.

[0025] The advantages or beneficial effects in the above technical solutions at least include:

[0026] The recombinant human type VII collagen of the present application can be efficiently obtained by recombining the codon-optimized nucleotide into a plasmid and expressing it in a host cell; and it has the collagen activity of promoting cell proliferation and promoting cell adhesion, can inhibit the expression of matrix metalloproteinase, has a cell repair effect, can be used as a cell repair product; and is widely used in food, cosmetics, health products or medical device products.

[0027] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the above-described illustrative aspects, embodiments and features, further aspects, embodiments and features of the present application will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In the drawings, unless otherwise specified, the same reference numerals throughout the several views denote the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0029] Figure 1 It is the plasmid map of the recombinant expression vector C7-pET-30b(+);

[0030] Figure 2 It is the map of the restriction enzyme digestion verification of the positive clone recombinant plasmid;

[0031] Figure 3 It is the expression electrophoresis map of the recombinant human type VII collagen before and after induction;

[0032] Figure 4It is the electrophoretogram of the expression of purified recombinant human type VII collagen;

[0033] Figure 5 It is the effect diagram of crystal violet staining of recombinant human type VII collagen promoting cell adhesion;

[0034] Figure 6 It is the diagram of the inhibitory effect of recombinant human type VII collagen on the expression level of MMP-9 gene;

[0035] Figure 7 It is the diagram of the inhibitory effect of recombinant human type VII collagen on the expression level of MMP-3 gene;

[0036] Figure 8 It is the diagram of the inhibitory effect of recombinant human type VII collagen on the expression level of MMP-1 gene. Detailed implementation manners

[0037] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0038] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources.

[0039] The technologies and procedures described or cited herein are conventional methods that are generally well-known and frequently used by those skilled in the art.

[0040] The present application provides a recombinant human type VII collagen, and the amino acid sequence of the recombinant human type VII collagen is as shown in SEQ ID NO: 1.

[0041] The present application also provides the nucleotide encoding the above-mentioned recombinant human type VII collagen; the nucleotide is as shown in SEQ ID No.2.

[0042] In the above specific implementation manners, nucleic acid molecules are the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecular compounds polymerized by many nucleotide monomers and are one of the most basic substances of life. The nucleotide sequence refers to the arrangement order of bases in DNA or RNA. The nucleic acid molecule contains cDNA. In some cases, the nucleic acid molecule can be modified for use in the vector of the present application, such as for codon optimization. In some cases, for the purpose of cloning into the vector, the sequence can be designed to contain terminal restriction site sequences. The nucleic acid molecule can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of the coding nucleic acid in one or more given cells or isolated from the one or more given cells.

[0043] The present application also provides an expression vector carrying the above nucleic acid molecule.

[0044] In a specific embodiment, an expression vector refers to a self-replicating DNA molecule that transfers a DNA fragment (target gene) to a recipient cell in recombinant DNA technology of genetic engineering. As the most commonly used and simplest vector in genetic engineering, it must include three parts: a genetic marker gene, a replication region, and a target gene. In addition to the commonly used Escherichia coli plasmid vectors, many other artificially constructed plasmid vectors applicable to microorganisms, yeasts, plants, etc. have been developed. Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends or no free ends (such as circular); nucleic acid molecules containing DNA, RNA or both; and other polynucleotide species known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell after being introduced into the host cell and thus replicate together with the host genome. In addition, some vectors are capable of directing the expression of a target gene. Such vectors are referred to herein as "expression vectors". A recombinant expression vector may contain a form suitable for expressing nucleic acids in a host cell, which means that the recombinant expression vector includes one or more regulatory elements that can be selected based on the host cell used for expression and that can be operably linked to the nucleic acid sequence to be expressed.

[0045] The present application also provides a host cell expressing the above recombinant human type VII collagen.

[0046] In a specific embodiment, a host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc. by a nucleic acid construct or expression vector containing the polynucleotide of the present application. "Host cell" encompasses any progeny of the parental cell that is not identical to the parental cell due to mutations occurring during the replication process. The host cell can be any cell useful in the production of recombinant human type VII collagen in the present application. To produce recombinant human type VII collagen, the nucleic acid encoding recombinant human type VII collagen can be isolated and inserted into one or more vectors for further cloning / or expression in the host cell. Such nucleic acid can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding recombinant collagen). The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, including primary transformed cells and their progeny, regardless of the number of passages. The progeny may not be completely identical to the parental cell in terms of nucleic acid content but may contain mutations. Methods for introducing vectors into host cells are well known, such as using electroporation to introduce the vector into the host cell, and the method can also be transfection, microinjection techniques, gene gun techniques, liposome-mediated methods, etc. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from Escherichia coli.

[0047] Example 1

[0048] Design of recombinant human type VII collagen

[0049] The nucleotide sequence of the nucleic acid molecule encoding the recombinant human type VII collagen of the present application is shown in SEQ ID NO.2.

[0050] SEQ ID NO.2 is:

[0051] atgggggataaaggagaccccggagtaggtctaccgggcccacgtggtgaacgcggcgaaccgggcatccgcggcgaagatggtcgtccgggccaagaaggtccgcgtggcttgaccggtcctccgggcagccgtggcgagcgcggtgagaagggtgacgttggtagcgctggactgaaaggtgacaagggcgacagcgctgtgattctgggcccaccgggtccgcgtggcgcgaaaggcgacatgggtgagcgcggtccgagaggcctggatggcgacaaaggtccgcgtggcgacaacggtgatccgggtgataagggctcgaaaggcgagccgggcgacaagggctctgcgggtctgccgggtctgcgtggcttgctgggtccacagggtcagccgggtgcggcaggcattccgggcgacccgggctccccgggtaaagatggtgttccggggatccgtggtgagaaaggtgacgtgggtttcatgggtccgcgtggtctcaagggtgagcgcggcgtcaaaggagcctgcggtttagatggtgaaaagggtgataagtaa。

[0052] Example 2. Construction of high-expression engineering bacteria and purification of recombinant human type VII collagen

[0053] This example provides a method for constructing high-expression engineering bacteria, including the following steps:

[0054] 1) Construction of an Escherichia coli recombinant expression vector:

[0055] Codon optimization was performed according to the Escherichia coli codon preference, and the obtained gene sequence was named C7; after adding the restriction enzyme sites NdeⅠ and XhoⅠ to both ends of the optimized sequence, total gene synthesis was carried out and cloned into the Escherichia coli expression vector pET-30b(+), obtaining the Escherichia coli recombinant expression vector C7-pET-30b(+)(the map is as Figure 1 shown).

[0056] 2) Plasmid transformation;

[0057] First, prepare competent Escherichia coli LTHINK(DE3):

[0058] ①Pick a single colony of Escherichia coli and inoculate it into 5 ml of LB liquid medium without antibiotics. Incubate it with shaking at 37 °C for 13 h until the late logarithmic growth phase. Inoculate the bacterial suspension into 100 ml of LB liquid medium at a ratio of 1:50 and incubate it with shaking at 37 °C for 2 - 3 h until OD600 = 0.5;

[0059] ②Transfer the culture broth into a centrifuge tube under sterile conditions, place it on ice for 10 min to cool the culture to 0 °C, and then centrifuge at 4 °C and 4000 rpm for 10 min;

[0060] ③Discard the supernatant and invert it for 1 min to drain the culture broth completely;

[0061] ④Gently suspend the cells in 10 ml of ice-precooled 0.1 mol / L CaCl 2 solution, mix well, place it on ice for 30 min, and then centrifuge at 4 °C and 4000 rpm for 10 min;

[0062] ⑤Discard the supernatant and invert it for 1 min to drain the last trace of culture broth completely;

[0063] ⑥Add 4 ml of ice-precooled 0.1 mol / L CaCl 2 solution containing 15% glycerol, gently suspend the cells, and place it on ice for a few minutes to obtain the competent cell suspension;

[0064] ⑦Aliquot 100 μL of Escherichia coli BL21(DE3) competent cells and store them at -70 °C for later use.

[0065] Transformation:

[0066] ①Take out a tube (100 μl) of LTHINK(DE3) competent bacteria from the -80 °C ultra-low temperature freezer, thaw it and place it on ice, and incubate it on ice for 5 - 10 min.

[0067] ②Add 50 ng of the constructed Escherichia coli recombinant expression vector C7-pET-30b(+), gently shake it and then place it on ice for 30 min.

[0068] ③Gently shake it well and then place it in a 42 °C water bath for 1 - 2 min for heat shock, and then quickly return it to ice and let it stand for 3 - 5 min.

[0069] ④Add 800 μl of LB medium (without antibiotics) to each of the above tubes in a laminar flow hood, gently mix it, and then fix it on the spring rack of the shaker and incubate it with shaking at 37 °C for 1 h.

[0070] ⑤Take 100 - 300 μl of the above transformation mixture in a laminar flow hood and drop it onto a solid LB plate culture dish containing the appropriate antibiotic, and spread it evenly with a glass spreader burned by an alcohol lamp.

[0071] ⑥ Mark the coated Petri dishes. First, place them in a 37 °C constant temperature incubator for 30 - 60 min until the liquid on the surface has penetrated into the culture medium, and then invert them and place them in the 37 °C constant temperature incubator overnight.

[0072] 3) Screening of positive clones:

[0073] Verify the recombinant quality of the recombinant positive clones by restriction enzyme mapping. The restriction enzymes used are MluI and XhoI, and the reaction system is shown in Table 1:

[0074] Table 1

[0075]

[0076] The reaction conditions are: 37 °C, reaction for 30 min. Detect by 1% agarose gel electrophoresis.

[0077] Reference Figure 2 , the recombinant positive clone recombinant plasmid can be verified, where Figure 2 1 in represents: DNA of the uncut recombinant expression vector C7-pET-30b(+); Figure 2 2 in represents: Plasmid digested by MluI and XhoI; Figure 2 M in represents: DNA Marker.

[0078] 4) Verification of PCR positive clones:

[0079] Inoculate the single colonies grown on the plate after overnight culture at 37 °C after transformation into 10 mL of LB liquid medium, add antibiotics, and culture overnight at 37 °C. And identify whether it is a positive clone by colony PCR. The colony PCR system is shown in Table 2 below:

[0080] Table 2

[0081] Component 20 μL System Forward primer(10 μmol / L) 0.5 μL Reverse primer(10 μmol / L) 0.5 μL <![CDATA[ddH 2 O]]> 8 μL Bacterial liquid 1 μL Taq DNA polymerase 10 μL TOTAL 20.0 μL

[0082] Forward primer: GATTAAAAAGGCGGCGAACAAA (SEQ ID NO.3);

[0083] Reverse primer: TCAAAGCCTTCTTTCGGGCT (SEQ ID NO.4).

[0084] The PCR program is as follows: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, 34 cycles; annealing at 50 - 54 °C for 30 s; extension at 72 °C for 30 s; final extension at 72 °C for 10 min; store at 4 °C. After PCR amplification, sequencing is performed. After comparison, it is consistent with the synthetic sequence.

[0085] 5) Expression of recombinant human type VII collagen:

[0086] Take a tube of stored bacteria from -80°C, thaw at room temperature, inoculate the above bacteria into 600 ml of LB culture medium containing 50 μg / ml Kan at a ratio of 1:1000, and shake at 220 rpm at 37°C until the OD of the bacteria 600 reaches 0.8 - 1.0; add inducer IPTG to a final concentration of 0.1 mM in 600 ml of fermentation medium (LB culture medium), and culture at 220 rpm at 37°C for 4 h and at 22°C for 8 h respectively; centrifuge at 5000 rpm at 4°C for 5 min, discard the supernatant and collect the fermented bacteria, and store at -20°C; for protein expression detection, break the bacteria, and the breaking conditions are: power of 150 W, break for 1 s, interval of 1 s, for a total of 15 min. Bacterial lysis buffer: TBS, pH 7.4.

[0087] Perform SDS-PAGE electrophoresis detection, and the results are as Figure 3 (shown in the expression of recombinant human type VII collagen), Figure 3 where M represents: Marker; 1 represents: before induction; 2 represents: whole bacteria after induction; 3 represents: supernatant after induction; 4 represents: precipitate after induction.

[0088] From Figure 3 it can be seen that after induced expression, recombinant human type VII collagen exists in both the bacteria and the supernatant, indicating that the recombinant human type VII collagen has been successfully expressed in the strain. After calculation, the expression level is 30%.

[0089] 6) Purification of recombinant human type VII collagen:

[0090] Bacterial cell disruption:

[0091] Resuspend the bacteria in lysis buffer (50 mM Tris-HCl) at a ratio of 1:10, perform high-pressure homogenization, once at 200 bar, twice at 800 bar, centrifuge at 9000 rpm at 4°C for 30 min. Collect the inclusion bodies.

[0092] Chromatographic purification:

[0093] Equilibrate a 5 mL strong cationic SP chromatography column with equilibration buffer (20 mM PB, pH 6.0), load the centrifuged supernatant at a flow rate of 2 mL / min, after loading, wash with equilibration buffer until the baseline is stable, and elute with elution buffer (20 mM PB, 300 mM NaCl, pH 6.0), and collect the elution peak during the elution process.

[0094] Perform SDS-PAGE electrophoresis detection, and the results are as Figure 4 (shown in the purification of recombinant human type VII collagen),Figure 4 M in the middle stands for: Marker; 1 stands for: after breaking the bacteria; 2 stands for: centrifugal supernatant; 3 and 4 stand for: sample penetration during the loading process; 5 stands for: after the balance solution is flushed and stabilized; 6 stands for: protein elution peak; 7 stands for: chromatography column cleaning peak.

[0095] from Figure 4 It can be seen that there is only recombinant human type VII collagen in the eluate, indicating that a high-purity collagen solution is obtained after purification.

[0096] Example 3: Recombinant human type VII collagen promotes cell proliferation

[0097] 1) Dissolving the sample to be tested: Filter the recombinant human type VII collagen (or existing collagen) to be tested with a 0.22 μm microporous filter membrane, and dilute it with 1×PBS in a gradient manner according to the concentrations of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.0625 mg / ml, 0.03125 mg / ml, 0.015 mg / ml, 0.008 mg / ml, and 0.004 mg / ml, and place it in a 4°C refrigerator for use.

[0098] 2) 3T3-L1 cell culture: 3T3-L1 cells were revived and inoculated into culture bottles. After the cells grew to 80-90%, they were subcultured and inoculated after the cells were in good growth condition.

[0099] 3) Collagen coating: Add 100 μL of standard solution, test sample, and PBS blank control to a 96-well plate. Prepare 4 wells for each sample coating and incubate at 37°C, 5% CO 2 After incubation in the incubator for 1 to 4 hours, remove excess coating solution from the wells.

[0100] 4) Cell proliferation assay: 3T3-L1 cells were cultured at 2×10 3 The cells were seeded into 96-well plates at a density of 100 cells / well and blank DMEM high-glucose medium was added to incubate in 5% CO 2 The cells were cultured at 37°C, and CCK8 detection reagent was added according to the instructions after 48 hours. After incubation for 2 hours, the cells were detected using an enzyme reader (450 nm wavelength).

[0101] 5) The experimental results showed that recombinant human type VII collagen protein could promote the proliferation of 3T3-L1 cells within the concentration range of 0.125 mg / mL to 1 mg / mL.

[0102] Example 4: Recombinant human type VII collagen promotes cell adhesion

[0103] 1) Dissolution of the sample to be tested: Recombinant human type VII collagen of the sample to be tested was filtered through a 0.22 μm microporous filter membrane and serially diluted with 1×PBS at concentrations of 1 mg / ml and 0.5 mg / ml, and then placed in a refrigerator at 4°C for standby.

[0104] 2) Coating preparation: Test articles and PBS blank controls were added to 96-well plates respectively. Four wells were prepared for each sample coating and incubated in an incubator at 37°C and 5% CO 2 for 1 h to 4 h. The excess coating solution was removed from the wells, and 100 μL of 1% BSA-PBS solution was added and incubated in an incubator at 37°C and 5% CO 2 for 1 h. After removing the liquid in the wells, they were washed three times with PBS, the washing solution was discarded, sealed with a sealing film and placed at 4°C for standby.

[0105] 4) Cell adhesion detection: 3T3-L1 cells were cultured in an incubator at 37°C and 5% CO 2 . The cell density and status were observed under an inverted microscope every day. When the cells grew to 80% - 90% of the culture flask, cell passage or cell seeding was carried out. 100 μL of cells were added to the wells at a density of 5×10 3 cells / well and incubated at 37°C and 5% CO 2 for 24 h.

[0106] The experimental results are as Figure 5 shown. As Figure 5 shown, according to the results of the 3T3-L1 cell adhesion experiment, recombinant human type VII collagen can promote the adhesion of 3T3-L1 cells within the concentration range of 0.5 mg / mL and 1 mg / mL.

[0107] Example 5: Recombinant human type VII collagen inhibits the expression of matrix metalloproteinases.

[0108] This example is related to the detection of the gene expression levels of MMP-9 (matrix metalloproteinase-9), MMP-3 (matrix metalloproteinase-3), and MMP-1 (matrix metalloproteinase-1).

[0109] The RT-PCR method was used to detect the gene transcription level in HSF cells. HSF cells in the logarithmic growth phase were seeded in six-well plates at a density of 3×10 5 cells / well. After the cells adhered for 24 h, 100 μg / mL and 50 μg / mL of recombinant human type VII collagen were used to act on HSF cells for 24 h respectively. Total cellular RNA was extracted by the TRIZOL method. 1 μg of RNA was taken and reverse transcription was carried out using the EasyScript 5*all-in (100 runs) AE341-02 kit. Using the synthesized cDNA by reverse transcription as a template, PCR reaction was carried out, and the reaction system was as shown in Table 3 below:

[0110] Table 3

[0111]

[0112]

[0113] The primer sequence design is shown in Table 4 below. The reaction conditions are as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s; annealing at 55°C for 30 s; extension at 72°C for 10 s, for a total of 40 cycles.

[0114] Table 4

[0115]

[0116] Using GAPDH as the reference gene, the calculation was performed using 2^-ΔΔct:. The results are as Figure 6 , Figure 7 , Figure 8 shown. It can be seen from Figures 6 - 8 that recombinant human type VII collagen can inhibit the expression of MMP-9, MMP-3, and MMP-1 genes and inhibit the degradation of the extracellular matrix.

[0117] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0118] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" can explicitly or implicitly include at least one of the features. In the description of the present application, "a plurality" means two or more, unless otherwise specifically defined.

[0119] The above is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application can easily think of various changes or substitutions, and these should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A recombinant human type VII collagen, characterized in that: The recombinant human type VII collagen comprises: a) the amino acid sequence shown in SEQ ID No.1; or b) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, and which retains the collagen activity of the amino acid sequence of SEQ ID NO.

1.

2. A nucleotide, characterized in that The nucleotide acid encodes the recombinant human type VII collagen according to claim 1.

3. A nucleotide according to claim 2, characterized in that The sequence of the nucleotide molecule is shown in SEQ ID NO:

2.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleotide according to claim 2 or 3.

5. The recombinant plasmid according to claim 4, characterized in that The plasmid vector is C7-pET-30b(+).

6. A highly efficient expression host cell, characterized in that: The highly efficient expression host cell comprises the nucleotide according to claim 2 or 3; or the recombinant plasmid according to claim 4 or 5.

7. The method for preparing recombinant human type VII collagen according to claim 1, characterized in that: The recombinant type III collagen is expressed using the high-efficiency expression host cell described in claim 6, and then separated and purified to obtain the recombinant human type VII collagen.

8. A cell repair reagent, characterized in that: The cell repair reagent contains the recombinant human type VII collagen according to claim 1; or the recombinant human type VII collagen encoded by the nucleotide according to claim 2 or 3; or the recombinant human type VII collagen expressed by the highly efficient host cell according to claim 6.

9. Use of the recombinant human type VII collagen according to claim 1, the recombinant human type VII collagen encoded by the nucleotide according to claim 2 or 3, or the recombinant human type VII collagen expressed by the highly efficient host cell according to claim 6 in the preparation of a cell repair reagent.

10. Use of the recombinant human type VII collagen according to claim 1, the recombinant human type VII collagen encoded by the nucleotides according to claim 2 or 3, or the recombinant human type VII collagen expressed by the highly efficient host cells according to claim 6 in food, cosmetics, health products or medical device products.

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