Recombinant type VII collagen Pro.C7 and its preparation method and application

Abstract

The present invention discloses a recombinant type VII collagen protein, Pro.C7, whose amino acid sequence is shown in any one of SEQ ID NOs. 1-8. Also disclosed are its encoding gene, expression vector, host bacteria, preparation method, and application. The present invention designs recombinant human type VII collagen based on a partial sequence of human type VII collagen. The protein is expressed in prokaryotes, and the expression system is mature. The purified protein has been experimentally verified to be non-cytotoxic. By organizing laminin, it promotes fibroblast migration and cytokine secretion, participates in skin damage and repair, and can be used as a biomaterial in cosmetic products, among other applications.

Description

Technical Field

[0001] The present invention relates to a recombinant type VII collagen Pro.C7 and a preparation method and application thereof, belonging to the technical field of protein expression. Background Art

[0002] Type VII collagen molecules assemble into anchoring fibrils, thick ribbons that extend from the basement membrane zone to the upper dermis. The presence of type VII collagen in the basement membrane has been confirmed by immunological methods and X-ray diffraction experiments. Type VII collagen consists of 2944 amino acids and is encoded by the COL7A1 gene. Type VII collagen fibrils are composed of three identical α-collagen chains, forming a homotrimer. They are composed of a central collagen domain flanked by the non-collagenous NC-1 and NC-2 domains. The central collagen domain exhibits the typical collagen gly-xy repeating structure. Flanked by the NC-1 and NC-2 non-collagenous domains at the N- and C-termini, it is localized to the basal zone beneath stratified squamous epithelial cells. The NC-1 domain: The N-terminal domain (NC-1) is a large globular region crucial for anchoring fibril formation. It participates in binding to other extracellular matrix components. NC-2 domain: The C-terminal domain (NC-2) is smaller and plays a role in the assembly of the triple helix and the stabilization of the collagen molecule.

[0003] Anchoring fibers composed of type VII collagen connect the epidermal basement membrane to the papillary dermis, holding the epidermis and dermis together and providing structure and stability. Traditionally, type VII collagen is isolated from the human placenta and purified using a multi-step process. This collagen extraction method has certain limitations, such as low extraction efficiency and low purity. With the development of genetic engineering technology, recombinant collagen has become the best alternative to animal-derived collagen in biomedicine and tissue engineering. Recombinant collagen is a protein obtained by cloning the human collagen gene into a selected expression vector, transforming it into expression cells, and finally purifying it. Compared with collagen obtained through traditional extraction, recombinant collagen has the advantages of being easier to process, free of viral risks, better water solubility, batch stability, and low rejection. Selecting the appropriate sequence is key to the industrialization of highly active recombinant collagen. Summary of the Invention

[0004] The purpose of the present invention is to provide a recombinant type VII collagen Pro.C7.

[0005] The technical solution adopted in the present invention is:

[0006] A recombinant type VII collagen Pro.C7, the amino acid sequence of which is shown in any one of SEQ ID NOs. 1-8.

[0007] The gene encoding the above-mentioned recombinant type VII collagen Pro.C7.

[0008] Preferably, the nucleotide sequence is shown in any one of SEQ ID NOs. 9-16.

[0009] The expression vector of the above-mentioned recombinant type VII collagen Pro.C7.

[0010] The host bacteria of the above-mentioned recombinant type VII collagen Pro.C7.

[0011] The preparation method of the recombinant type VII collagen Pro.C7 comprises the following steps:

[0012] (1) constructing the above-mentioned expression vector and transforming it into the expression host bacteria;

[0013] (2) Cultivating the expression host bacteria to induce the expression of the recombinant protein;

[0014] (3) Purify the expression product to obtain the above-mentioned recombinant type VII collagen Pro.C7.

[0015] The application of the above-mentioned recombinant type VII collagen Pro.C7 in promoting cell proliferation in vitro.

[0016] The application of the above-mentioned recombinant type VII collagen Pro.C7 in promoting cell migration in vitro.

[0017] The above-mentioned recombinant type VII collagen Pro.C7 is used to promote cell secretion of elastin in vitro.

[0018] The use of the above-mentioned recombinant type VII collagen Pro.C7 in the preparation of skin care products.

[0019] Beneficial effects of the present invention:

[0020] The present invention designs recombinant human type VII collagen based on a partial sequence in human type VII collagen. It is expressed by prokaryotes and the expression system is mature. The purified protein has been experimentally verified to be non-cytotoxic. It promotes fibroblast migration and cytokine secretion by organizing laminin, participates in skin damage and repair, and can be used as a biomaterial in skin care products and other beauty products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Plasmid map of the recombinant expression vector pET30a-C7-1 of the present invention.

[0022] Figure 2The electrophoresis results of the recombinant collagen protein of the present invention, the lanes from left to right are: Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Marker, Pro.C7-5, Pro.C7-6, Pro.C7-7, Pro.C7-8.

[0023] Figure 3 Preliminary cytotoxicity experimental results of the recombinant collagen proteins Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, and Pro.C7-8 of the present invention.

[0024] Figure 4 Cell scratch test results of the recombinant collagen proteins Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, and Pro.C7-8 of the present invention.

[0025] Figure 5 Elastin ELISA test results of the recombinant collagen proteins Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, and Pro.C7-8 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further illustrated by the following examples, but is not intended to limit the present invention. The specific materials and their sources used in the embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Example 1: Preparation of recombinant type VII collagen Pro.C7 plasmids

[0028] (1) Gene design and synthesis

[0029] First, an expression vector of recombinant human type VII collagen was constructed, wherein the recombinant type VII collagen Pro.C7 were ro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, and Pro.C7-8, and their corresponding amino acid sequences were SEQ ID No.1-8, and their corresponding coding gene sequences were SEQ ID No.9-16. The corresponding coding genes were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0030] (2) Construction of recombinant expression vector

[0031] The above-synthesized gene fragment was used as a template for PCR amplification, and the target gene fragment and the pET30a expression vector backbone were connected by Gibson-plasmid ligation to obtain the corresponding plasmid.

[0032] The primer sequences are shown in Table 1:

[0033] Table 1 Primer sequences

[0034]

[0035]

[0036] Table 2 PCR amplification system

[0037] System components Ingredient volume Primer F 2.5 μL Primer R 2.5 μL 2xSuperNova PCR Mix(Dye) 25 μL Encoding genes for commissioned biosynthesis of Qingke 1 μL <![CDATA[ddH2O]]> To 50μL

[0038] After preparing the PCR system shown in Table 2, mix thoroughly and centrifuge. PCR amplification conditions were as follows: initial denaturation at 98°C for 30 seconds; second stage: denaturation at 98°C for 10 seconds, annealing at 50-72°C for 30 seconds, and extension at 72°C for 30 seconds / kb, for 33 cycles; third stage: final extension at 72°C for 2 minutes. The vector and gene fragment were recovered using a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) according to the product instructions.

[0039] Table 3 Gibson connection system

[0040]

[0041]

[0042] After mixing the above components on ice, place them in a 37°C heat bath for 60 min. The obtained ligation products were stored on ice or at -20°C for subsequent competent cell transformation.

[0043] The ligation products were transformed into the host bacteria E. coli DH5α by heat shock method, spread on LB culture resistance plates, and incubated at 37°C overnight. Positive clones were randomly picked and cultured in LB liquid medium at 37°C, 220 rpm overnight. Plasmids were extracted using a plasmid rapid extraction kit. The successful plasmids pET30a-C7-1, pET30a-C7-2, pET30a-C7-3, pET30a-C7-4, pET30a-C7-5, pET30a-C7-6, pET30a-C7-7, and pET30a-C7-8 were constructed. Their maps can be found in Figure 1 .

[0044] (3) Construction of engineered bacteria

[0045] The recombinant expression plasmid obtained above was transferred into Escherichia coli competent cells BL21 (DE3) by heat shock, and positive Escherichia coli genetically engineered bacteria were screened. The specific process was as follows: ① Take 5 μL of the recombinant expression plasmid into 100 μL of Escherichia coli competent cells BL21 (DE3) and let it stand on ice for 30 minutes; ② Heat shock the mixture in a 42°C water bath for 90 seconds, and then quickly put it on ice and let it stand for 2 minutes; ③ Add 500 μL of non-resistant LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the mixture, and culture it at 37°C, 220 rpm for 0.5 hours; ④ Take 200 μL of the bacterial solution and evenly spread it on the surface of the mixture. 5. Incubate the plate upside down in a 37°C incubator for about 16 hours until clearly visible colonies appear, and obtain the corresponding DE3-pET30a-C4-1, DE3-pET30a-C4-2, DE3-pET30a-C4-3, DE3-pET30a-C4-4, DE3-pET30a-C4-5, DE3-pET30a-C4-6, DE3-pET30a-C4-7, and DE3-pET30a-C4-8 engineered bacteria.

[0046] Example 2: Induced expression of engineered bacteria

[0047] Place a single colony from the plate in LB medium containing ampicillin and incubate at 37°C, 220 rpm, for 10 hours. This serves as the primary seed solution. Inoculate a 2% inoculum into fresh LB medium and incubate at 37°C for 2 hours. Induce expression by adding IPTG to a final concentration of 0.2 mM at 18°C ​​for 20 hours. Collect the cells by centrifugation at 4000g, 4°C, for 20 minutes.

[0048] The cells were resuspended in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, pH 8.5), 100X protease inhibitor PMSF was added, and then the cells were disrupted by ultrasonication at 360W for 30 min (ultrasonication for 3 s with a 6 s interval). 1 mL of the disrupted liquid was centrifuged at 4°C and 10,000 g for 10 min, 80 μL of the supernatant was taken, and the precipitate was resuspended with 200 μL of lysis buffer. 120 μL of the resuspension was discarded, and 20 μL of 5X protein loading buffer was added to both the supernatant and the precipitate. After mixing, the mixture was heated in a boiling water bath for 10 min for SDS-PAGE.

[0049] Example 3: Purification of expression products

[0050] The disrupted liquid obtained in Example 2 was centrifuged at 10,000 g and 4° C. for 30 minutes. The supernatant was collected and washed with water on a Ni affinity column. The column was equilibrated with buffer 1 (25 mM Tris, 200 mM NaCl, pH 8.0) and loaded with the sample. Contaminants were rinsed with a wash buffer containing 20 mM imidazole (20 mM imidazole, 25 mM Tris, 200 mM NaCl, pH 8.0). The target protein was eluted with a solution containing 250 mM imidazole (250 mM imidazole, 25 mM Tris, 200 mM NaCl, pH 8.0). The column was washed with a solution containing 1 M imidazole and then with water. Finally, the column was filled with 20% ethanol.

[0051] After purification, 80 μL of supernatant was taken and 20 μL of 5X protein loading buffer was added. After mixing, the mixture was heated in a boiling water bath for 10 min and then subjected to SDS-PAGE. It can be seen that Pro.C7-1 to 8 are all soluble. Figure 2 shown.

[0052] Example 4: Bioactivity Detection of Recombinant Humanized Type VII Collagen

[0053] Cytotoxicity and cell scratch experiments were performed using the recombinant type VII collagen Pro.C7 obtained in Example 3

[0054] (1) Preliminary experiment (cytotoxicity experiment)

[0055] Cytotoxicity test. Use ultrapure water to dissolve the protein sample, and the protein content is expressed as mass volume (W / V). The results of the cytotoxicity test are as follows: Figure 3 As shown, it can be seen that the prepared recombinant type VII collagen Pro.C7 has no cytotoxicity and can promote cell proliferation.

[0056] (2) Formal experiment (cell scratch test)

[0057] Add 2 mL of diluted cell suspension to a 6-well plate to make the concentration 1×10 6 cells / well. After cells have adhered and filled approximately 80-90% of the 6-well plate, pretreat with DMEM medium containing mitomycin C for 3 hours. Scratch the plate with a 10μL pipette tip, rinse three times with PBS, then add DMEM medium containing mitomycin C and the sample protein solution to a final protein concentration of 1.0%. After adding the sample, place the plate under an inverted microscope to photograph and record the scratch width at 0 hours, then place it in an incubator and incubate for 24 hours. After 24 hours, remove the 6-well plate from the CO2 incubator and place it under an inverted microscope to photograph and record the scratch width at 24 hours.

[0058] Calculation: Scratch healing rate (%) = (S0 scratch area - S 24 Scratch area) / S0 scratch area×100%.

[0059] The results are as follows Figure 4 As shown, in this experiment, the sample group within the detection concentration range had significant differences compared with the control group (blank control), and had the effect of promoting cell migration.

[0060] (3) Compactness experiment

[0061] The effect of recombinant type VII collagen Pro.C7 on elastin levels in fibroblasts was analyzed using an Elastin ELISA kit (Human Elastin ELISA Kit, Abcam) according to the manufacturer's instructions. Elastin levels are expressed as mean ± SD. Specific assay procedures can be adjusted according to the kit's instructions.

[0062] Take a sterile ELISA plate, design blank wells, standard wells, and sample wells to be tested respectively, add 50 μL of standards of different concentrations to each standard well, add 10 μL of the sample to be tested to the sample well, wherein the sample to be tested is a 5.0% protein solution, and then add 40 μL of sample diluent (i.e., the sample is diluted 5 times), and nothing is added to the blank wells; except for the blank wells, add 100 μL of HRP-labeled detection antibody to each standard well and sample well, seal the reaction wells with a sealing film, incubate at 37°C in the dark for 60 min, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 min, discard the washing solution, pat dry on absorbent paper, repeat this washing 5 times, add 50 μL of substrate A and B to each well, incubate at 37°C in the dark for 15 min, add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0063] like Figure 5As shown, recombinant humanized type VII collagen Pro.C7 has a unique advantage in elastin production, with the highest elastin content in fibroblasts in the sample group reaching 1721.11 pg / mL. This further demonstrates that recombinant humanized type VII collagen Pro.C7 can increase skin firmness and firmness by promoting the production of large amounts of elastin by cells.

Claims

1. A recombinant type VII collagen Pro.C7, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the recombinant type VII collagen Pro.C7 according to claim 1.

3. The coding gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

11. The expression vector of the recombinant type VII collagen Pro.C7 according to claim 1 .

5. The host bacteria of the recombinant type VII collagen Pro.C7 according to claim 1.

6. The method for preparing the recombinant type VII collagen Pro.C7 according to claim 1, characterized in that The steps include: (1) Constructing the expression vector described in claim 4 and transforming it into an expression host bacterium; (2) Cultivate the expression host bacteria and induce the expression of the recombinant protein; (3) Purify the expression product to obtain the recombinant type VII collagen Pro.C7 described in claim 1. 7 . Use of the recombinant type VII collagen Pro.C7 according to claim 1 in promoting cell proliferation in vitro, wherein the cells are HaCaT cells.

8. Use of the recombinant type VII collagen Pro.C7 according to claim 1 in promoting cell migration in vitro, wherein the cells are HaCaT cells.

9. Use of the recombinant type VII collagen Pro.C7 according to claim 1 in promoting elastin secretion by cells in vitro, wherein the cells are fibroblasts.

10. Use of the recombinant type VII collagen Pro.C7 according to claim 1 in the preparation of skin care products.

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