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

Abstract

The invention belongs to the technical field of optimized coding genes, and discloses a recombinant human V-type collagen as well as a preparation method and application thereof, the amino acid sequence of the recombinant human V-type collagen comprises a basic repetitive unit, and the basic repetitive unit is VQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHR. The collagen provided by the invention has no cytotoxicity, has good effects of promoting cell proliferation and cell migration, is high in safety, and has the potential of being applied to the fields of medicines, medical instruments, biological materials and tissue engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of optimized coding genes, and in particular relates to a recombinant human type V collagen and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant protein in mammalian tissues, significantly impacting the normal function and repair of cells, tissues, and even organs. At least 28 types of collagen have been discovered, and based on their structural characteristics, collagen is classified into fibroblastic collagen, fibroblast-associated collagen (FACIT collagen), lattice-forming collagen, and membrane collagen. Types I, II, and III collagen are the most common, accounting for 80% to 90% of human collagen. Type V collagen is less abundant in the human body, comprising only 2% to 5% of human collagen. It is primarily found in bone, dermis, cornea, and placenta, and can be used for wound healing and scar prevention.

[0003] Currently, collagen can be divided into animal collagen and recombinant collagen. Animal collagen is primarily derived from terrestrial and marine animals, but the extraction and purification requirements for animal collagen are stringent, with issues such as limited raw material sources, the risk of viral transmission, and poor batch-to-batch consistency. With the advancement of genetic engineering technology, research is increasingly focusing on recombinant collagen production. Recombinant collagen production effectively avoids the problems associated with animal collagen. Furthermore, recombinant collagen has a single molecule and a clear structure, making the separation and purification process relatively simple. Furthermore, the product is highly pure and safe.

[0004] Nowadays, type I, type II and type III collagen are widely used in tissue repair, cartilage repair and facial filling. The applicant has conducted research in this regard and obtained CN114671946B, a recombinant human type III collagen, its preparation method and application, and CN118359701A, a recombinant human type II collagen, its preparation method and application.

[0005] Type V collagen, a tissue-specific collagen matrix, plays a crucial role in promoting cell proliferation and migration. In medicine and cosmetics, it can promote wound healing, joint and bone health, improve fine lines, and inhibit melanin production. Therefore, identifying a recombinant type V collagen that is bioactive and suitable for industrialization is crucial. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects in the prior art and provide a recombinant human type V collagen having the effects of promoting cell proliferation and cell migration, as well as a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is: Technical Topic 1 A recombinant human type V collagen, the amino acid sequence of which includes a basic repeating unit, wherein the basic repeating unit is: VQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHR.

[0008] As a further improvement of the present invention, the basic repeating unit is repeated 6 times, and its amino acid sequence is shown as SEQ ID No.1.

[0009] As a further improvement of the present invention, the nucleotide sequence of the recombinant human type V collagen is shown as SEQ ID No. 2.

[0010] Technical Topic 2 A vector comprising the nucleotide sequence shown in SEQ ID No. 2.

[0011] Technical Theme 3 A host cell, comprising the vector described in the second technical topic.

[0012] Technical Theme 4 A method for preparing the recombinant human type V collagen described in the first preparation technology theme specifically comprises the following steps: (1) Protein sequence design Based on human type V collagen, the amino acid sequence shown in SEQ ID No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type V collagen in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type V collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type V collagen fragment; (3) Construction of expression vector: The nucleic acid fragment obtained in step (2) was ligated to the pET30a(+) plasmid via the NdeI and KpnI multiple cloning sites to obtain an expression plasmid; (4) Construction and screening of expression strains: The plasmid described in step (3) is transferred into the competent host cell by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Induce the expression strain obtained in step (4) and collect the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human type V collagen.

[0013] As a further improvement of the present invention, the host cell is Escherichia coli BL21 (DE3).

[0014] Technical Topic 5 A use of the recombinant human type V collagen as described in technical theme 1 in the preparation of pharmaceutical products, medical devices, biomaterials, and tissue engineering.

[0015] The beneficial effects of adopting the above technical solution are: (1) Experimental results show that the recombinant human type V collagen provided by the present invention has a good ability to promote cell migration and cell proliferation.

[0016] (2) The recombinant human type V collagen provided by the present invention has no cytotoxicity and no animal-derived infection source, has high biosafety, and has the prospect of being applied in the fields of medicine, medical devices, biomaterials, tissue engineering, cosmetics, etc.

[0017] (3) The preparation method of the recombinant human type V collagen of the present invention is simple and suitable for industrial large-scale production, thus solving the problem of limited source of type V collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an electrophoresis diagram of a sample of the affinity chromatography process of recombinant human type V collagen HC115 prepared in Example 5. In the diagram: 1: Marker, 2: Whole bacteria, 3: Supernatant filtration, 4: 1000 mL flow-through, 5: 1800 mL flow-through, 6: 2600 mL flow-through, 7: 40% elution, 8: 100% elution, 9: BSA; Figure 2 This is an electrophoresis diagram of a sample of the recombinant human type V collagen HC115 prepared in Example 5 during cation exchange chromatography. In the diagram: 1: Marker, 2: affinity chromatography sample, 3: flow-through 300 mL, 4: flow-through 300 mL, 5: flow-through 600 mL, 6: 50% elution, 7: 100% elution, 8: desalting, 9: BSA. Figure 3 This is a statistical chart of the cytotoxicity experimental data of recombinant human type V collagen HC115 at different concentrations in Effect Example 1; Figure 4 This is the statistical chart of the data of the cell proliferation promotion test of recombinant human type V collagen HC115 in effect example 2. *** indicates P < 0.001 compared with the blank group; Figure 5 This is the scratch area diagram of effect example 3: recombinant human type V collagen HC115 promotes cell migration; Figure 6 This is a statistical chart of the data from the cell migration promotion test of recombinant human type V collagen HC115 in effect example 3. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0020] Example 1 The accession number of human type V collagen was retrieved from UniprotKB database: P20908; then, https: / / blast.ncbi.nlm.nih.gov / The online tool blastb was used to blast P20908, and collagen sequences from different species similar to P20908 were obtained. The sequence IDs and sources are shown in the table below: After obtaining the amino acid sequences of the above proteins, motif analysis was performed using the online tool https: / / meme-suite.org / meme to obtain conserved sequences across different species. The conserved sequences whose amino acid sequences are shown below were selected for subsequent experiments: VQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHR The above conserved sequence was taken and repeated 6 times, with the N-terminus starting with methionine to reduce the degradation rate of the protein; at the same time, a histidine tag was added to facilitate identification and purification, resulting in the collagen sequence HC115, whose amino acid sequence is shown in SEQ ID NO.1.

[0021] SEQ ID NO.1 MVQGPPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRVQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRVQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRVQG PPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRVQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRVQGPPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHRHHHHHH.

[0022] The coding nucleic acid sequence was reverse-designed using the online design tool Jcat (http: / / www.jcat.de / ) and codon-optimized for expression in the host E. coli. Following this optimization, a nucleic acid fragment encoding recombinant human type V collagen was obtained. The nucleotide sequence corresponding to the amino acid sequence is shown in SEQ ID NO. 2: SEQ ID NO.2 CATATGGTACAAGGTCCTCCAGGACCCGCTGGGAAACCGGGTCGTCGCGGTCGCGCGGGTTCGGACGGCGCGCGTGGTATGCCGGGACAAACCGGTCCGAAGGGCGACCGTGGCTTCGATGGCTTGGCAGGCCTGCCGGGTGAGAAAGGTCATCGTGTTCAGGGTCCTCCGGGGCCAGCGGGCAAACCGGGCCGCCGTGGACGGGCAGGTAGCGACGGTGCGAGAGGTATGCCGGGTCAGACTGGTCCGAAGGGTGATCGTGGATTCGACGGTCTTGCCGGTCTGCCGGGTGAAAAGGGCCACCGTGTTCAGGGTCCGCCAGGTCCGGCGGGTAAGCCGGGCCGCCGTGGCCGTGCGGGCTCCGACGGCGCTCGTGGCATGCCTGGGCAAACCGGCCCCAAGGGTGACCGTGGTTTTGATGGTCTGGCGGGTCTGCCGGGCGAGAAAGGTCACCGCGTGCAAGGTCCGCCGGGCCCGGCTGGCAAGCCGGGCCGTCGCGGTCGCGCGGGCTCTGACGGCGCACGTGGCATGCCGGGTCAGACCGGTCCGAAAGGCGATCGCGGTTTTGATGGTTTGGCGGGCCTGCCAGGTGAAAAAGGCCACCGTGTCCAGGGTCCTCCGGGTCCGGCAGGCAAGCCGGGCCGTCGTGGTCGTGCGGGCAGCGATGGTGCTCGCGGTATGCCGGGCCAAACCGGTCCGAAAGGCGACCGTGGTTTTGATGGCCTGGCCGGATTGCCGGGCGAGAAGGGTCACCGCGTGCAGGGCCCACCGGGTCCGGCTGGCAAACCGGGCCGTAGAGGCCGTGCCGGTAGCGATGGTGCCCGTGGTATGCCGGGTCAAACGGGTCCGAAGGGCGACCGCGGATTCGACGGTCTGGCGGGCTTACCGGGTGAAAAAGGACATCGACATCACCACCACCATCATTAAGGTACC (2) Gene synthesis: Based on the nucleic acid sequence shown above, a nucleic acid fragment (HC115) encoding a recombinant human type V collagen fragment was synthesized by GenScript Biotech Co., Ltd.

[0023] Example 2 Construction of expression vector pET30a(+)-HC115 The nucleic acid fragment HC115 obtained in Example 1 was ligated into the pET30a(+) plasmid via the NdeI and KpnI multiple cloning sites to obtain the recombinant plasmid pET30a(+)-HC115.

[0024] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-HC115 Pick a single colony of E. coli BL21 (DE3) and inoculate it into an LB tube, then shake and culture it overnight at 37°C; add 0.5 mL of the overnight culture to a 50 mL LB flask, shake vigorously at 37°C for about 2 hours to allow the bacteria to grow to the early logarithmic phase; aseptically transfer the bacteria to an ice-cold 50 mL polypropylene tube and place it on ice for 10 minutes; centrifuge at 4°C, 4000 rpm, pour off the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible; add 6 mL of ice-cold 0.1 mol / L CaCl2 to resuspend the pellet and place it on ice for 30 minutes; centrifuge at 4°C, 3000 rpm, pour off the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible; add 1.2 mL of ice-cold 0.1 mol / L CaCl2 to resuspend the pellet (if you want to prepare competent cells for storage at -70°C, add 0.1 mol / L containing 20% glycerol). CaCl2 suspension of bacterial cells), incubated at 4°C for 5-24 hours, 200 µL of BL21 (DE3) competent cell suspension was aspirated, and the recombinant plasmid pET30a(+)-HC115 prepared in Example 2 (volume <10 µL, DNA <50 ng) was added to the competent cells, gently mixed, and incubated on ice for 30 minutes; then, static heat shock was performed in a 42°C water bath for 90 seconds, and the cells were immediately cooled on ice; 500 µL of liquid LB culture medium was added, mixed, and the cells were revived in a shaker at 37°C for 45 minutes; the transformed cells were aspirated and spread on a plate supplemented with antibiotics (kanamycin), and the plate was incubated upside down in a 37°C incubator. The colonies that grew were the expression strain BL21 (DE3) / pET30a (+) -HC115.

[0025] Example 4 Induced expression of expression strain BL21(DE3) / pET30a(+)-HC115 A single colony of the expression strain BL21(DE3) / pET30a(+)-HC115 prepared in Example 3 was picked and incubated in LB liquid medium containing 50 μg / mL Kan. After overnight cultivation at 37°C and 200 rpm, the colony was activated and then inoculated into a 5 L fermenter containing 3 L of complete medium at a 3% inoculum size. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. The fermentation was continued until OD 600 When the expression reached 60, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce expression, and the culture was continued for 12 hours, and the cells were collected by centrifugation.

[0026] Example 5 Purification of recombinant human type V collagen HC115 The theoretical isoelectric point of HC115 is 11.66, and the pH of the buffer solution is 7.5. The specific purification steps are as follows: 1. Bacteria destruction The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) at a cell to cell ratio (m / v) of 1:5 to 1:20, with 1:10 selected in this example. The cells were disrupted using a high-pressure homogenizer. The disrupted bacterial solution was centrifuged at 12,000 g for 1 hour, and the supernatant was collected and filtered through a 0.45 µm filter.

[0027] 2. Affinity chromatography (1) Column balance: The Ni Sepharose FF column was equilibrated with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) until the baseline was stable.

[0028] (2) Loading: The supernatant was loaded onto the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0029] (3) Column flushing: Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0030] (4) Target protein elution: Elution was performed with 60% Buffer A and 40% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type V collagen HC115.

[0031] 3. Ion exchange chromatography (1) Sample processing: The affinity chromatography eluate was diluted with ion exchange solution A (20 mM Tris, pH 7.5) or ultrafiltered to a conductivity of less than 5 mS / cm.

[0032] (2) Column balance: The SP Sepharose FF column was equilibrated with buffer A until the baseline was stable.

[0033] (3) Loading: The refolding solution was loaded onto the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0034] (4) Column flushing: Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0035] (5) Target protein elution: Elution was performed with 50% ion exchange solution A and 50% ion exchange solution B (20 mM Tris, 1 M NaCl, pH 7.5), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type V collagen HC115.

[0036] 4. Desalination The HC115 solution obtained by ion exchange chromatography was replaced with purified water by equal volume replacement using a 5 kDa ultrafiltration membrane device to remove salt from the solution, and then filtered with a 0.22 µm filter membrane and freeze-dried to obtain a recombinant collagen freeze-dried product.

[0037] Example 6 SDS-PAGE protein electrophoresis detection Sample Preparation: Collect the bacterial cells prepared in Example 4 and the purified sample from Example 5, add loading buffer, mix thoroughly, boil in a boiling water bath for 10 minutes, and cool naturally before use. Load the samples onto a GenScript SurePAGE™ precast gel (4-12%) and run electrophoresis at 140V for 45-55 minutes until the bromophenol blue band reaches the bottom of the gel.

[0038] Microwave staining with Coomassie Brilliant Blue R-250: 1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R-250 to a final concentration of 0.1% (w / v) in 40% ethanol and 10% acetic acid. 2) Prepare the destaining solution: Dissolve 10% (v / v) ethanol and 7.5% (v / v) acetic acid to a final concentration. 3) After electrophoresis, remove the gel from the slides and place it in a staining container containing 100 mL of staining solution. 4) Cover the container and microwave on high for 8 minutes. To avoid any risk, be careful not to allow the solution to boil. 5) Remove the staining container from the microwave and gently shake it on a destaining shaker at room temperature for 5 minutes. 6) Discard the staining solution and carefully rinse the gel with deionized water. 7) Discard the deionized water and add 100 mL of destaining solution. 8) Cover the container and microwave on high for 8 minutes. 9) Pour off the destaining solution, add new destaining solution, and repeat step 8; 10) Remove from the microwave oven and place on a destaining shaker at room temperature and gently shake until the background is clear.

[0039] The results of protein electrophoresis test are shown in the attached Figure 1 and attached Figure 2 As shown, it proves that recombinant human type V collagen HC115 was successfully prepared and separated.

[0040] Effect Example 1 Cytotoxicity Test of Recombinant Human Type V Collagen HC115 The cytotoxicity of HC115 was evaluated according to the method specified in GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test MTT Cytotoxicity Test. The experiment was divided into two groups: the sample group (recombinant human type V collagen HC115 solution prepared in Example 5), the positive control group (100µL complete MEM medium containing 0.2g / mL ZDEC), and the blank / negative control group (100µL complete MEM medium). L929 cells were seeded in 96-well plates for 24 hours and then treated with the buffer or cell culture medium described in each group. After 24 hours, if the negative control group and the positive control group met the requirements, the relative cell viability was detected using MTT. Figure 3 As shown in the figure, HC115 had no obvious cytotoxicity in the concentration range of 10 μg / mL~800 μg / mL.

[0041] Effect Example 2: Recombinant human type V collagen HC115 promotes cell proliferation test The cell proliferation test was conducted using the recombinant human type V collagen HC115 prepared in Example 5. The main experimental process was as follows: human skin keratinocytes HaCaT were cultured at 1×10 4100μL complete DMEM medium was used to dissolve 10, 50, 100, 500, and 800 μg / mL HC115 samples. Each well was inoculated into a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. The culture medium in the wells was discarded, and a sample group (100μL complete DMEM medium containing 10, 50, 100, 500, and 800 μg / mL HC115 samples) and a control group (100μL complete DMEM medium) were set up. Five replicate wells were set up in each group for sample treatment. After 24 hours of culture, the supernatant was discarded, the MTT solution was added and shaken, and the plate was placed in an incubator for 2 hours. After incubation, DMSO was added and shaken for 15 minutes, and the absorbance was detected in a microplate reader. Cell viability (%) = (OD of the experimental group - OD of the blank group) / (OD of the control group - OD of the blank group) * 100%. Figure 4 As shown in the figure, the experimental results showed that compared with the control group, recombinant human type V collagen had a significant proliferation-promoting effect at concentrations of 10-800 μg / mL.

[0042] Effect Example 3: Recombinant human type V collagen HC115 promotes cell migration test This effect example was performed in accordance with the "Pharmaceutical Industry Standard of the People's Republic of China" YY / T1849-2022 Recombinant Collagen Appendix C: Cell Migration Test-Cell Scratch Method.

[0043] The cell migration test was carried out using the recombinant human type V collagen HC115 prepared in Example 5. The main experimental process was as follows: human skin keratinocytes HaCaT were cultured at 1×10 6 Cells were seeded in 6-well plates, and 2 mL of culture medium was added to each well. The cells were cultured at 37°C and 5% CO₂ for 24 hours. After 24 hours of culture, a 10 µL pipette tip was aligned vertically with a ruler against the well plate (a scratch-resistant cell culture dish could also be used) and gently pushed downward to create a scratch along the vertical (or horizontal) line. The cells were rinsed three times with PBS to remove the scratched cells. Sample groups (HC115 samples dissolved in DMEM serum-free medium) and controls (DMEN serum-free medium) were set up. Sample groups were supplemented with 2 mL of serum-free medium at concentrations of 10, 50, 100, 500, and 800 µg / mL, respectively. The control group was treated with serum-free medium alone. The cells were cultured at 37°C and 5% CO2, and photographed under a 40x microscope at the intersection of the horizontal and vertical scratch lines at 0, 24, 48, and 72 hours. Nine images were obtained for each well, and 27 data points were collected for each group. Cell migration rate (%) = (initial scratch area - scratch area at time t) / initial scratch area. Figure 5 and Figure 6 As shown in the figure, the experimental results showed that compared with the control group, recombinant human type V collagen had a significant migration-promoting effect at concentrations of 10-800 μg / mL.

[0044] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human type V collagen, characterized in that: Its amino acid sequence includes a basic repeating unit, and the basic repeating unit is: VQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGEKGHR.

2. The recombinant human type V collagen according to claim 1, characterized in that The basic repeating unit is repeated 6 times, and its amino acid sequence is shown in SEQ ID No.

1.

3. The recombinant human type V collagen according to claim 2, characterized in that: The nucleotide sequence of the recombinant human type V collagen is shown in SEQ ID No.

2.

4. A carrier, characterized in that The vector contains the nucleotide sequence shown in SEQ ID No.

2.

5. A host cell, characterized in that The host cell contains the vector according to claim 4.

6. A method for preparing recombinant human type V collagen according to claim 1, characterized in that: The specific steps include: (1) Protein sequence design Based on human type V collagen, the amino acid sequence shown in SEQ ID No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human type V collagen in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type V collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type V collagen fragment; (3) Construction of expression vector: The nucleic acid fragment obtained in step (2) was ligated to the pET30a(+) plasmid via the NdeI and KpnI multiple cloning sites to obtain an expression plasmid; (4) Construction and screening of expression strains: The plasmid described in step (3) is transferred into the competent host cell by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Induce the expression strain obtained in step (4) and collect the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption and centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human type V collagen.

7. The preparation method according to claim 6, characterized in that The host cell is Escherichia coli BL21 (DE3).

8. Use of the recombinant human type V collagen according to claim 1 in the preparation of pharmaceutical products, medical devices, biomaterials, and tissue engineering.

Images