Recombinant VII type collagen Pro.C7 as well as preparation method and application thereof

Abstract

The invention discloses a recombinant VII type collagen Pro.C7. The amino acid sequence of the recombinant VII type collagen Pro.C7 is shown as any one of SEQ ID NO.1-8. The invention also discloses a coding gene, an expression vector, host bacteria, a preparation method and application of the gene. The recombinant human VII type collagen is designed on the basis of a part of sequences in the human VII type collagen, prokaryote expression, mature expression system and purified protein have no cytotoxicity through experimental verification, fibrocyte migration and cytokine secretion are promoted through tissue laminin, and the human VII type collagen participates in skin injury and repair, so that the human VII type collagen can be used for repairing skin. The material can be applied to beauty products and the like as a biological material.

Description

Technical Field

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

[0002] Type VII collagen molecules assemble into anchoring fibrils, which are thick ribbon-like structures extending 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 fibers are composed of three identical alpha collagen chains, forming a homotrimer. It consists of a central collagen domain and non-collagen NC-1 and NC-2 domains on both sides. The central collagen domain presents a typical collagen gly-x-y repeat sequence structure. The NC-1 and NC-2 non-collagen domains are on both sides of the N-terminal and C-terminal, and are confined to the basal region under the stratified squamous epithelial cells. NC-1 domain: The N-terminal domain (NC-1) is a large globular region, which is crucial for the formation of anchoring fibrils. It participates in the 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 collagen molecules.

[0003] The anchoring fibers composed of type VII collagen connect the epidermal basement membrane to the papillary dermis, keeping the epidermal and dermal layers of the skin together and providing structure and stability. Traditionally, type VII collagen was isolated from human placenta and purified using a multi-step process. This method of collagen extraction has certain limitations, such as low extraction efficiency and low purity. With the development of genetic engineering technology, recombinant collagen has become the best substitute for animal-derived collagen in biomedicine and tissue engineering. Recombinant collagen is obtained by cloning the human collagen gene into a selected expression vector, transforming it into expression cells, and finally through purification technology. Compared with the collagen obtained by traditional extraction, recombinant collagen mainly has the advantages of processability, no virus risk, good water solubility, batch stability and low rejection reaction. Selecting the appropriate sequence is the key to the industrialization of highly active recombinant collagen. Summary of the Invention

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

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

[0006] A recombinant type VII collagen Pro.C7, whose amino acid sequence is shown in any one of SEQ ID NO.1-8.

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

[0008] Preferably, its nucleotide sequence is as shown in any one of SEQ ID NO.9-16.

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

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

[0011] The preparation method of the above-mentioned recombinant type VII collagen Pro.C7, the steps of which include:

[0012] (1) Construct the above-mentioned expression vector and transform it into an expression host bacterium;

[0013] (2) Culture the expression host bacterium 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 application of the above-mentioned recombinant type VII collagen Pro.C7 in promoting the secretion of elastin by cells in vitro.

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

[0019] The beneficial effects of the present invention:

[0020] Based on a partial sequence of human type VII collagen, the present invention designs recombinant human type VII collagen, which is expressed by prokaryotes. The expression system is mature, and the purified protein has been experimentally verified to be non-cytotoxic. Through tissue laminin, it promotes the migration and secretion of cytokines by fibroblasts and participates in the injury and repair of the skin. It can be used as a biomaterial in beauty products such as skin care products. Description of the Drawings

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

[0022] Figure 2Electrophoresis results of the recombinant collagen protein of the present invention. From left to right in the lanes 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 test results of the recombinant collagen Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, Pro.C7-8 of the present invention.

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

[0025] Figure 5 Elastin ELISA test results of the recombinant collagen Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, Pro.C7-8 of the present invention. Detailed implementation mode

[0026] The present invention will be further illustrated by the following examples, which shall not be construed as limiting the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials of the same or similar type, model, quality, nature or function as the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

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

[0028] (1) Gene design and synthesis

[0029] First, construct an expression vector for recombinant human type VII collagen. Among them, the recombinant type VII collagen Pro.C7 are Pro.C7-1, Pro.C7-2, Pro.C7-3, Pro.C7-4, Pro.C7-5, Pro.C7-6, Pro.C7-7, Pro.C7-8 respectively. Their corresponding amino acid sequences are SEQ ID No.1-8, and the corresponding coding gene sequences are SEQ ID No.9-16. The corresponding coding genes were entrusted to Beijing Tsingke Biotechnology Co., Ltd. for synthesis.

[0030] (2) Construction of the recombinant expression vector

[0031] Using the synthesized gene fragment as a template, perform PCR amplification, and ligate the target gene fragment and the pET30a expression vector backbone by Gibson-plasmid ligation method 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 Component volume Primer F 2.5 μL Primer R 2.5 μL 2xSuperNova PCR Mix (Dye) 25 μL Coding gene synthesized by Tsingke Biological 1 μL <![CDATA[ddH 2 O]]> To 50 μL

[0038] After preparing the PCR system shown in Table 2, mix well and centrifuge. The PCR amplification conditions are: the first stage is pre-denaturation at 98°C for 30 s; the second stage is denaturation at 98°C for 10 s, annealing at 50-72°C for 30 s, extension at 72°C for 30 s / kb, with 33 cycles; the third stage is final extension at 72°C for 2 min. Use a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) to recover the above vector and gene fragment, and operate according to the operation steps of the product manual.

[0039] Table 3 Gibson ligation system

[0040]

[0041]

[0042] Mix the above components on ice and then place them in a 37°C water bath for 60 min. Store the obtained ligation product on ice or at -20°C for subsequent competent cell transformation.

[0043] The ligation product was transformed into the host bacterium E. coli DH5α by heat shock method, spread on an LB culture resistant plate, incubated overnight at 37°C. Positive clones were randomly picked and cultured overnight in LB liquid medium at 37°C and 220 rpm. Plasmids were extracted using a plasmid rapid extraction kit. The successfully constructed plasmids pET30a-C7-1, pET30a-C7-2, pET30a-C7-3, pET30a-C7-4, pET30a-C7-5, pET30a-C7-6, pET30a-C7-7, pET30a-C7-8, and their maps are shown in Figure 1 .

[0044] (3) Construction of engineered bacteria

[0045] The above-obtained recombinant expression plasmids were transferred into Escherichia coli competent cells BL21(DE3) by heat shock method, and positive Escherichia coli genetically engineered bacteria were screened. The specific process was as follows: ① Take 5 μL of the recombinant expression plasmid and add it to 100 μL of Escherichia coli competent cells BL21(DE3), and let it stand on ice for 30 min; ② Heat shock the mixture in a 42°C water bath for 90 s, and then quickly place it on ice for 2 min; ③ Add 500 μL of antibiotic-free 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 and 220 rpm for 0.5 h; ④ Take 200 μL of the bacterial solution and evenly spread it on an LB solid medium plate containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / mL ampicillin); ⑤ Invert the plate and culture it in a 37°C incubator for about 16 h until clear colonies grow, and obtain the corresponding engineered bacteria 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, DE3-pET30a-C4-8.

[0046] Example 2: Induced expression of engineered bacteria

[0047] The single colonies on the above plate were placed in LB liquid medium containing ampicillin antibiotic, cultured at 37°C and 220 rpm for 10 hours. This was the primary seed liquid, which was inoculated into a new LB medium at an inoculation amount of 2%, cultured at 37°C for 2 h, added with IPTG at a final concentration of 0.2 mM, and induced expression at 18°C for 20 hours. The cells were collected by centrifugation at 4000 g and 4°C for 20 min.

[0048] The cell pellet was 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 lysed by sonication with a 360 W ultrasonic device for 30 min (sonication for 3 s with a 6 s interval). After that, 1 mL of the lysate was centrifuged at 4 °C and 10,000 g for 10 min. 80 μL of the supernatant was taken, the pellet was resuspended in 200 μL of lysis buffer, 120 μL of the resuspended solution was discarded, and 20 μL of 5X protein loading buffer was added to both the supernatant and the pellet. After mixing, they were heated in a boiling water bath for 10 min for SDS-PAGE.

[0049] Example 3: Purification of the expression product

[0050] The lysate obtained in Example 2 was centrifuged at 10,000 g and 4 °C for 30 min, and the supernatant was collected. The Ni affinity column material was washed with deionized water and equilibrated with buffer 1 (25 mM Tris, 200 mM NaCl, pH 8.0). The sample was loaded, and the column was rinsed with a washing buffer containing 20 mM imidazole (20 mM imidazole, 25 mM Tris, 200 mM NaCl, pH 8.0) to remove impurities, and 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 material was washed with a solution containing 1 M imidazole, then with deionized water, and finally filled with 20% ethanol.

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

[0052] Example 4: Bioactivity detection of recombinant humanized collagen type VII

[0053] The recombinant collagen type VII Pro.C7 obtained in Example 3 was used for cytotoxicity and cell scratch assays

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

[0055] Cytotoxicity detection. The protein sample was dissolved in ultrapure water, and the protein content was expressed as mass / volume (W / V). The results of the cytotoxicity assay are as Figure 3 shown. It can be seen that the prepared recombinant collagen type VII Pro.C7 has no cytotoxicity and can promote cell proliferation.

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

[0057] Add 2 mL of the diluted cell suspension to a 6-well plate to make the concentration 1×10 6 cells / well. After the cells adhere and grow to about 80 - 90% confluence in the 6-well plate, pretreat them with DMEM medium containing mitomycin C for 3 h. Scratch with a 10 μL pipette tip, wash three times with PBS, then add DMEM medium containing mitomycin C and the sample protein solution to make the final concentration of the protein 1.0%. After adding the samples, place them under an inverted microscope, take a picture of the scratch width at 0 h and record it, and then place them in an incubator for 24 h. After 24 h, take out the 6-well plate from the CO 2 incubator, place it under an inverted microscope, take a picture of the scratch width at 24 h and record the data.

[0058] Calculation: Scratch healing rate (%) = (S 0 scratch area at 0 h - S 24 scratch area at 24 h) / S 0 scratch area at 0 h × 100%.

[0059] Results are as Figure 4 shown. In this experiment, within the detected concentration range, there were significant differences between the sample group and the Control group (blank control), indicating that it has the effect of promoting cell migration.

[0060] (3) Tightening experiment

[0061] Use the operation manual of the Elastin ELISA kit (Human Elastin ELISA Kit, Abcam) to detect and analyze the effect of recombinant type VII collagen Pro.C7 on the content of Elastin in fibroblasts. The results of Elastin content are expressed as Mean ± SD. The specific detection steps can be routinely adjusted according to the kit manual.

[0062] Take a sterile ELISA plate, and design blank wells, standard wells, and wells for the samples to be tested. Add 50 μL of standards with different concentrations to each standard well, add 10 μL of the sample to be tested to the sample wells. The sample to be tested is a protein solution with a concentration of 5.0%, and then add 40 μL of sample diluent (i.e., the sample is diluted 5 times), and do not add anything to the blank wells. Except for the blank wells, add 100 μL of the detection antibody labeled with HRP 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, and repeat the washing process 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] As Figure 5As shown, recombinant humanized type VII collagen Pro.C7 has a unique advantage in elastin production. The elastin content in fibroblasts in the sample group can reach up to 1721.11 pg / mL at most. This further demonstrates that recombinant humanized type VII collagen Pro.C7 can increase skin tightness by promoting cells to produce a large amount of elastin, making the skin firm.

Claims

1. A recombinant type VII collagen Pro.C7, characterized in that Its amino acid sequence is shown in any one of SEQ ID NO.1-8.

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 The nucleotide sequence thereof is shown in any one of SEQ ID NOs.9-16.

4. 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 as claimed in 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 according to claim 4 and transforming it into an expression host bacterium; (2) Cultivating the expression host bacteria and inducing the expression of the recombinant protein; (3) Purify the expression product to obtain the recombinant type VII collagen Pro.C7 as described in claim 1.

7. Use of the recombinant type VII collagen Pro.C7 according to claim 1 in promoting cell proliferation in vitro.

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

9. Use of the recombinant type VII collagen Pro.C7 according to claim 1 in promoting cell secretion of elastin in vitro.

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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