A recombinant human type IV collagen, its preparation method and application

Abstract

The present invention belongs to the technical field of optimized coding gene, and discloses a recombinant human type IV collagen, a preparation method and an application thereof. The amino acid sequence thereof is shown as SEQ ID No.1. The collagen provided by the present invention has been verified by experiments to have a high yield, good cell proliferation promoting performance, no cytotoxicity, high safety, and can be widely applied in the fields of medicine, medical devices, biological materials, tissue engineering and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of optimized coding gene, and particularly relates to a recombinant human type IV collagen, a preparation method thereof and an application thereof. Background Art

[0002] Collagen is the most abundant protein in the body, the main structural protein of human tissues and organs, accounting for about 25%-35% of the total human protein, and also the main component of the extracellular matrix (ECM), accounting for about 85% of the extracellular matrix; it is commonly found in skin, blood vessels, tendons, fascia and other parts, and plays an important role in maintaining the normal physiological functions of cells, tissues and organs and in injury repair. In recent years, due to its biocompatibility and low antigenicity that are far beyond those of other high molecular materials, collagen is regarded as a safe biomedical material and has been widely used in the pharmaceutical, health care product and cosmetic industries. So far, there are at least 28 different types of collagen, which are located in different tissues and organs respectively.

[0003] Members of the collagen family have a unique triple-helix structure. According to whether they can form collagen fibrils with periodic cross striations, they are divided into fibrillar collagen and nonfibrillar collagen. Common fibrillar collagens include type I, II, III, V and XI collagens, etc., and common nonfibrillar collagens include type IV, X collagens, etc. Among them, type IV collagen mainly exists in the basement membrane and plays an important role in the formation of the extracellular matrix and the regulation of cell functions. Type IV collagen not only provides a scaffold for cell growth, but also can recognize and interact with specific receptors on the cell surface, thereby activating intracellular signal transduction pathways and participating in important physiological processes such as cell adhesion, migration, growth, proliferation and differentiation. The lack of type IV collagen will lead to abnormal structure and function of the basement membrane, and thus affect the normal physiological activities of cells.

[0004] At present, the sources of collagen mainly include extraction from animal tissues and expression through genetic engineering technology. Type IV collagen has a low content in animals. Traditionally, type IV collagen is isolated from human placentas and purified using a multi-step process. This method of collagen extraction has certain limitations, such as low extraction efficiency and low purity. Recombinant collagen mainly has the advantages of processability, no virus risk, good water solubility, batch stability, and low rejection reaction. Therefore, using genetic engineering technology to produce type IV collagen is bound to be the best choice. Unfortunately, some problems have been encountered: The recombinant expression of the full-length type IV collagen has not been successful. Although some truncated proteins of type IV collagen can be recombinantly expressed, their yields are not high and / or their biological activities are much worse than those of the full-length type IV collagen. Therefore, they are not suitable for industrial-scale production (as disclosed in CN 117903287 A).

[0005] In view of this, there is a need in the art for a recombinantly expressed human type IV collagen with good stability and capable of being prepared in large quantities in an industrial system. Summary of the Invention

[0006] The object of the present invention is to overcome the defects existing in the prior art, and to provide a recombinantly expressed human type IV collagen that can be industrially produced and has good stability, and at the same time provide its preparation method and application.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] On the one hand, the present invention provides a recombinantly expressed human type IV collagen, and the recombinant collagen includes one or more of the following characteristics:

[0009] (1) Its amino acid sequence is as shown in SEQ ID No.1, specifically as follows:

[0010] MGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATEGPKGDRGPQGQPGLPGLPGPMGPPGLPGIDGVKGDKGNPGWPGAPGVPGPKGDPGFQGMPGIGGSPGITGSKGDMGPPGVPGFQGPKGLPGLQGIKGDQGDQGHHHHHH

[0011] (2) Its amino acid sequence is an amino acid sequence modified by substituting, deleting, or adding one or several amino acids on the basis of SEQ ID No.1;

[0012] (3) An amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID No: 1.

[0013] On the other hand, the present invention provides a nucleic acid encoding the above-mentioned recombinant human type IV collagen, and the nucleotide sequence of the recombinant human type IV collagen comprises one or more of the following characteristics:

[0014] (1) Its nucleotide sequence is as shown in SEQ ID No. 2, specifically as follows:

[0015] ATGGGCGTTAAAGGCGAAGCCGGTTTACCGGGCACCCCGGGTCCGACGGGCCCGGCGGGCCAAAAGGGCGAACCGGGCAGCGATGGCATCCCGGGCAGCGCGGGCGAAAAAGGCGAACCGGGCTTACCGGGCCGCGGTTTTCCGGGCTTCCCGGGCGCGAAAGGCGATAAAGGTAGCAAAGGCGAGGTTGGCTTTCCGGGTCTGGCGGGCAGTCCGGGCATTCCGGGTAGCAAAGGTGAACAAGGCTTCATGGGTCCACCGGGTCCGCAAGGTCAGCCGGGCTTACCGGGCAGTCCGGGCCATGCCACGGAGGGCCCAAAAGGCGATCGCGGCCCACAAGGCCAACCGGGCTTACCGGGTCTGCCGGGTCCGATGGGTCCACCGGGCCTGCCGGGCATTGATGGCGTGAAAGGTGACAAAGGTAACCCGGGTTGGCCGGGCGCGCCGGGCGTGCCGGGCCCGAAAGGCGATCCGGGTTTTCAAGGCATGCCGGGCATCGGCGGCAGCCCGGGCATTACCGGCAGCAAAGGCGATATGGGTCCGCCGGGCGTTCCGGGCTTTCAAGGCCCGAAAGGCTTACCGGGCTTACAAGGCATTAAAGGCGATCAAGGCGATCAAGGCCATCACCATCACCATCATTAA

[0016] (2) Any nucleotide sequence obtained by synonymous codon substitution of (1).

[0017] On yet another aspect, the present invention provides a vector containing any one or more of the above-mentioned nucleic acids.

[0018] In another aspect, the present invention provides a host cell containing the above-mentioned vector.

[0019] In another aspect, the present invention provides a method for preparing the above-mentioned recombinant human type IV collagen, specifically including the following steps:

[0020] (1) Protein sequence design

[0021] Based on human type IV collagen, an amino acid sequence as shown in SEQ No.1 is designed;

[0022] (2) Gene design and synthesis

[0023] According to the amino acid sequence of the recombinant human type IV collagen described in step (1), the encoding nucleic acid sequence is designed in reverse and codon-optimized to obtain a nucleotide sequence encoding the recombinant human type IV collagen, and then gene synthesis is carried out to obtain a nucleic acid fragment encoding the recombinant human type IV collagen fragment;

[0024] (3) Construction of expression vector:

[0025] It is ligated to the pET30a(+) plasmid through the NdeI and XhoI multiple cloning sites to obtain an expression plasmid;

[0026] (4) Construction and screening of expression strains:

[0027] The plasmid described in step (3) is transferred into the host cell competent state by heat shock method, coated on a resistance plate containing kanamycin, and cultured to obtain an expression strain;

[0028] (5) Induced expression

[0029] The expression strain obtained in step (4) is induced to express, and the bacterial liquid is collected;

[0030] (6) Purification

[0031] The bacterial liquid collected in step (5) is subjected to cell disruption, centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human type IV collagen.

[0032] As some preferred embodiments of the present invention, the host cell is Escherichia coli BL21(DE3).

[0033] In another aspect, the present invention provides the use of the above-mentioned recombinant human type IV collagen in cosmetics, pharmaceuticals, health products, medical devices or biological materials.

[0034] The beneficial effects produced by adopting the above technical solutions are as follows:

[0035] (1)The recombinant human type IV collagen provided by the present invention can be expressed in an Escherichia coli expression system with high expression yield, and is suitable for large-scale industrial production.

[0036] (2)The recombinant human type IV collagen provided by the present invention has been verified by experiments to have good effects on promoting cell migration and proliferation, and has good safety and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the following will briefly introduce the drawings required for use in the description of the specific embodiments.

[0038] Figure 1 Electrophoresis diagram of samples during the affinity chromatography process of the recombinant human type IV collagen HC84 prepared in Example 5;

[0039] Figure 2 Electrophoresis diagram of samples during the ion chromatography process of the recombinant human type IV collagen HC84 prepared in Example 5;

[0040] Figure 3 Statistical chart of cytotoxicity experiment data of recombinant human type IV collagen HC84 at different concentrations in Effect Example 1;

[0041] Figure 4 Statistical chart of data on the promotion of cell proliferation by recombinant human type IV collagen HC84 in Effect Example 2;

[0042] Figure 5 Statistical chart of data on the promotion of cell migration by recombinant human type IV collagen HC84 in Effect Example 3;

[0043] Figure 6 Statistical chart of data on the cell adhesion test of recombinant human type IV collagen HC84 in Effect Example 4;

[0044] Figure 7 Electrophoresis diagram of samples in the cell stability test of recombinant human type IV collagen HC84 in Effect Example 5; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following will clearly and completely describe the invention in combination with specific embodiments.

[0046] Example 1 Gene Design and Synthesis

[0047] (1) Gene Design:

[0048] According to the sequence characteristics of human type IV collagen, the collagen sequence HC84 was designed. Its amino acid sequence is shown in SEQ ID NO.1. It starts with methionine at the N-terminus to reduce the protein degradation rate, and a histidine tag is added at the C-terminus for easy identification and purification. The amino acid sequence of the recombinant human type IV collagen fragment is as follows:

[0049] SEQ ID NO.1

[0050] MGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATEGPKGDRGPQGQPGLPGLPGPMGPPGLPGIDGVKGDKGNPGWPGAPGVPGPKGDPGFQGMPGIGGSPGITGSKGDMGPPGVPGFQGPKGLPGLQGIKGDQGDQG HHHHHH

[0051] Using the online design tool Jcat (http: / / www.jcat.de / ), its coding nucleic acid sequence was reverse-designed, and codon optimization was performed for expression in the host Escherichia coli. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human type IV collagen was obtained. The nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2:

[0052] ATGGGCGTTAAAGGCGAAGCCGGTTTACCGGGCACCCCGGGTCCGACGGGCCCGGCGGGCCAAAAGGGCGAACCGGGCAGCGATGGCATCCCGGGCAGCGCGGGCGAAAAAGGCGAACCGGGCTTACCGGGCCGCGGTTTTCCGGGCTTCCCGGGCGCGAAAGGCGATAAAGGTAGCAAAGGCGAGGTTGGCTTTCCGGGTCTGGCGGGCAGTCCGGGCATTCCGGGTAGCAAAGGTGAACAAGGCTTCATGGGTCCACCGGGTCCGCAAGGTCAGCCGGGCTTACCGGGCAGTCCGGGCCATGCCACGGAGGGCCCAAAAGGCGATCGCGGCCCACAAGGCCAACCGGGCTTACCGGGTCTGCCGGGTCCGATGGGTCCACCGGGCCTGCCGGGCATTGATGGCGTGAAAGGTGACAAAGGTAACCCGGGTTGGCCGGGCGCGCCGGGCGTGCCGGGCCCGAAAGGCGATCCGGGTTTTCAAGGCATGCCGGGCATCGGCGGCAGCCCGGGCATTACCGGCAGCAAAGGCGATATGGGTCCGCCGGGCGTTCCGGGCTTTCAAGGCCCGAAAGGCTTACCGGGCTTACAAGGCATTAAAGGCGATCAAGGCGATCAAGGCCATCACCATCACCATCATTAA

[0053] (2)Gene synthesis:

[0054] According to the nucleic acid sequence shown above, a nucleic acid fragment encoding a recombinant human type IV collagen fragment was synthesized by GenScript Biotech Corporation.

[0055] Example 2 Construction of expression vector pET30a(+)-HC84

[0056] The nucleic acid fragment SEQ ID NO.2 obtained in Example 1 (through the NdeI and XhoI multiple cloning sites) was ligated to the pET30a(+) plasmid to obtain the recombinant plasmid pET30a(+)-HC84.

[0057] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-HC84

[0058] The construction of the expression strain was carried out with reference to the method described in "Molecular Cloning: A Laboratory Manual (Third Edition)" (written by J. Sambrook et al.). The specific steps are as follows: Pick a single colony of Escherichia coli BL21(DE3) and inoculate it into an LB test tube, then culture it overnight with shaking at 37°C; Add 0.5 ml of the overnight culture to a 50-ml Erlenmeyer flask containing LB, and culture it vigorously with shaking at 37°C for about 2 hours until the bacteria grow to the early logarithmic phase; Transfer the bacteria to a 50-ml polypropylene tube pre-cooled with ice under sterile conditions, and place it on ice for 10 minutes; Centrifuge at 4°C and 4000 rpm, pour out the supernatant, invert the tube to allow as much residual liquid to flow out as possible; Add 6 ml of ice-precooled 0.1 mol / L CaCl2 to resuspend the pellet, and place it on ice for 30 minutes; Centrifuge at 4°C and 3000 rpm, pour out the supernatant, invert the tube to allow as much residual liquid to flow out as possible; Add 1.2 ml of ice-precooled 0.1 mol / L CaCl2 to resuspend the pellet (if preparing competent cells for storage at -70°C for later use, add 0.1 mol / L CaCl2 containing 20% glycerol to suspend the bacteria), place it at 4°C for 5 - 24 hours, then pipette 200 μl of the competent cell suspension, add the recombinant plasmid pET30a(+)-HC84 prepared in Example 2 (volume < 10 μl, DNA < 50 ng), mix gently, and place it on ice for 30 minutes; Heat shock at 42°C in a water bath for 90 seconds with static incubation, and immediately cool it on ice; Add 500 μl of liquid LB medium, mix well, and place it on a shaker at 37°C for low-speed shaking for 45 minutes to recover (or directly place it in a 37°C water bath for 1 hour after adding LB, and shake the tube in the middle to suspend the cells); Pipette the transformed cells and spread them on a plate containing antibiotics (kanamycin), and incubate it inverted in a 37°C incubator. The grown colonies are the expression strain BL21(DE3) / pET30a(+)-HC84.

[0059] Example 4 Induced Expression of the Expression Strain BL21(DE3) / pET30a(+)-HC84

[0060] Pick a single colony of the expression strain BL21(DE3) / pET30a(+)-HC84 prepared in Example 3 and inoculate it into an LB liquid medium containing 50 μg / mL Kan. Culture it overnight at 37°C and 200 r / min to obtain an activated seed, and then inoculate it into a 5-L fermenter containing 3 L of CM medium at an inoculation amount of 3%. Control the temperature at 37°C, the dissolved oxygen at 30%, and the pH at 7.0 during the fermentation process. When the OD 600 reaches 60, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.5 mM for induced expression, and continue to culture for 12 hours. Then centrifuge to collect the bacterial cells.

[0061] Example 5 Purification of Recombinant Human Type IV Collagen HC84

[0062] The theoretical isoelectric point of HC84 is 7.07, and the theoretical molecular weight is 20.04 kDa. The purification of recombinant human type IV collagen was carried out using a Ni affinity chromatography column and an ion exchange chromatography column. The specific purification steps are as follows:

[0063] 1. Bacterial cell disruption

[0064] 1 kg of bacterial cells prepared in Example 4 was resuspended with an appropriate amount of cell disruption solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.5). The ratio of bacterial cells to cell disruption solution (m / v) was 1:10 - 1:20, and 1:10 was selected in this example. The bacterial cells were disrupted using a high-pressure homogenizer. The disrupted bacterial solution was centrifuged at 12,000 g for 1 h, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane.

[0065] 2. Affinity chromatography

[0066] (1) Column equilibration:

[0067] The Ni Sepharose FF chromatography column was equilibrated with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.5) until the baseline was stable.

[0068] (2) Sample loading:

[0069] The supernatant was loaded onto the chromatography column, and the column retention time was controlled to be not less than 5 min.

[0070] (3) Column washing:

[0071] The chromatography column was washed with buffer A until the baseline was stable, not less than 7 CVs.

[0072] (4) Elution of the target protein:

[0073] Elution was carried out with 80% Buffer A and 20% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8.5), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type IV collagen HC84.

[0074] 3. Ion exchange chromatography

[0075] (1) Sample treatment:

[0076] The eluate from affinity chromatography was diluted with buffer C (20 mM Tris, pH 5.5) or ultrafiltered and replaced until the conductivity was less than 5 mS / cm.

[0077] (2) Column equilibration:

[0078] Equilibrate the SP Chromstar FF chromatography column with buffer C for 5 column volumes until the baseline is stable.

[0079] (3) Sample loading:

[0080] Load the displacement solution onto the chromatography column and control the column retention time to be no less than 5 min.

[0081] (4) Column washing:

[0082] Wash the chromatography column with buffer C until the baseline is stable, no less than 7 column volumes.

[0083] (5) Elution of target protein:

[0084] Elute with 60% Buffer C and 40% Buffer D (20 mM Tris, 1 M NaCl, pH 5.5), collect the eluate into a new centrifuge tube to obtain a solution containing recombinant human type IV collagen HC84.

[0085] 4. Desalting

[0086] Replace the HC84 solution obtained in step 3 with ultrapure water using a 5 kDa ultrafiltration membrane device by equal-volume displacement to remove the salt in the solution, and lyophilize to obtain the recombinant collagen lyophilized product. 20 g of lyophilized product can be obtained from 1 kg of bacterial cells.

[0087] Example 6 SDS-PAGE protein electrophoresis detection

[0088] Sample treatment: Collect the samples from the affinity chromatography process and the ion chromatography process in Example 5, add loading buffer and mix evenly, heat in a metal bath at 100 °C for 5 min, and cool naturally for standby. Use the GenScript SurePAGE™ precast gel (4-12%) for sample loading and electrophorese at 140 V for 45-55 minutes until the bromophenol blue band runs to the bottom of the gel.

[0089] Coomassie Brilliant Blue R-250 Staining Using a Microwave Oven: (1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R250 at a final concentration of 0.1% (W / V) in a solution of 40% ethanol and 10% acetic acid; (2) Prepare the decolorizing solution: Dissolve ethanol at a final concentration of 10% (V / V) and acetic acid at 7.5% (V / V) together; (3) After electrophoresis, pry open the gel plate to remove the gel, and then place it in a staining container containing 100 ml of the staining solution; (4) Cover the container lid and place it in the microwave oven to heat at high power for 8 minutes. To avoid danger, note that the solution should not be allowed to boil; (5) Remove the staining container from the microwave oven and gently shake it on a decolorizing shaker at room temperature for 5 minutes; (6) Pour out the staining solution and carefully wash the gel with deionized water; (7) Pour out the deionized water and add 100 ml of the decolorizing solution; (8) Cover the lid, place it in the microwave oven and heat at high power for 8 minutes; (9) Pour out the decolorizing solution, add fresh decolorizing solution, and repeat step 8; (10) Remove it from the microwave oven and gently shake it on a decolorizing shaker at room temperature until the background is clear.

[0090] The result diagram is as attached Figure 1 and 2 shown.

[0091] Effect Example 1 Cytotoxicity Experiment of Recombinant Human Type IV Collagen HC84

[0092] Evaluate the cytotoxicity of HC84 according to the method specified in GB / T 16886.5-2017 Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Test - Appendix C MTT Cytotoxicity Test. The experiment was divided into four groups: a sample group (recombinant human type IV collagen HC84 solution prepared in Example 5), a positive control group (extract of ZDEC Polyurethane Film), a negative control group (extract of High Density Polyethylene Film), and a medium control group (cell culture medium containing 10% serum, placed at 37 ± 1°C, 24 ± 2 h). L929 cells were cultured in the extracts or cell culture media of each group. When the negative control group and the positive control group met the requirements, calculate the relative cell proliferation rate. As Figure 3 shown, HC84 has no obvious cytotoxicity in the concentration range of 0.1 mg / ml to 10 mg / ml.

[0093] Effect Example 2 Cell Proliferation Promotion Experiment of Recombinant Human Type IV Collagen HC84

[0094] HaCaT skin keratinocytes at 5×10 3Cells were inoculated into the wells. After inoculating the cells in the 96-well culture plate, it was placed in an incubator at 37 °C and 5% CO2 and cultured for 24 h, 48 h, and 72 h respectively. The culture medium in the wells was discarded. In the blank control group, 100 μL of culture medium was added to each well, and in the sample group, 100 μL of HC84 solution was added to each well. The 96-well culture plate was placed in an incubator at 37 °C and 5% CO2 and cultured for 24 h, 48 h, and 72 h. 10 μL of CCK8 reagent was added to each well and incubated in the incubator for 2 h. After incubation, the absorbance value at a wavelength of 450 nm of each well was measured using a microplate reader. As Figure 4 shown, recombinant human type IV collagen HC84 has a good cell proliferation effect.

[0095] Effect Example 3: Cell migration promotion test of recombinant human type IV collagen HC84

[0096] According to the "Standard for the Pharmaceutical Industry of the People's Republic of China" YY / T 1849-2022 Recombinant Collagen Appendix C: Cell Migration Test - Cell Scratch Method, evaluate the effect of recombinant humanized type IV collagen HC84 on the migration rate of HaCaT skin keratinocytes. Digest the cells and count them. Inoculate 5×10 5 HaCaT skin keratinocytes into each well of the six-well plate, and the total volume of each well is 2 mL. Culture at 37 °C and 5% CO2 for 24 hours. Use a 200 μL pipette tip to align vertically with a ruler and gently push down to form scratches longitudinally (transversely). Rinse the cells 3 times with PBS to remove the scratched cells; in the sample group (add 2, 4, 6, 8, 10 mg / ml HC84), add 2 ml of the sample prepared with serum-free medium, and the blank control group only adds serum-free medium. Take pictures under the microscope at 0 h, 24 h, 48 h, and 72 h, and calculate the cell migration rate of each group. As Figure 5 shown, compared with the control group, HC84 significantly induced the migration of HaCaT cells and had a time-dependent effect.

[0097] Effect Example 4: Cell adhesion test of recombinant human type IV collagen HC84

[0098] Digest HaCaT skin keratinocytes and count them. Inoculate 1×10 5HaCaT keratinocytes, 100 μL / well. The blank control group (BC) was added with only PBS, and the sample groups (added with 2, 4, 6, 8, 10 mg / ml HC84), and the positive control group (PC, the control group with the maximum enzyme activity of the sample). Discard the culture medium, and wash each sample once with PBS buffer. Treat for 24 h (5% CO2, 37 °C, >90% humidity). One hour before the scheduled detection time, use the LDH release reagent to treat the "control well with the maximum enzyme activity of the sample". The addition amount was 10% of the original culture medium volume. After adding the LDH release reagent, pipette and mix well, and continue to incubate. At the scheduled time, centrifuge the cell culture plate at 400 g for 5 min using a multi-well centrifuge. Take 120 μL of the supernatant and transfer it to a new 96-well plate. Add 60 μL of the LDH detection solution to each well, mix well, and incubate in the dark at room temperature for 30 min. Measure the absorbance at 490 nm. The results are as Figure 6 shown. Compared with the control group, HC84 at concentrations of 2 mg / ml and 4 mg / ml had the ability to increase the level of LDH secreted by HaCaT cells, thereby enhancing cell adhesion.

[0099] Effect Example 5 Stability test of recombinant human type IV collagen HC84

[0100] Take 10 mg of the same batch of freeze-dried product samples stored at room temperature for 3, 6, and 12 months respectively, dissolve them in 1 ml of ultrapure water and mix well, and perform SDS-PAGE protein electrophoresis detection. As Figure 7 shown, recombinant human type IV collagen HC84 has good stability.

[0101] The above examples provide a recombinant human type IV collagen that can achieve industrial production. Verified by the effect examples, this recombinant human type IV collagen has no cytotoxicity and can promote the proliferation of L929 mouse fibroblasts.

[0102] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that it is still possible to modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human type IV collagen, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. A nucleic acid encoding the recombinant human type IV collagen according to claim 1, characterized in that, The nucleotide sequence of the recombinant human type IV collagen is shown in SEQ ID No.

2.

3. A carrier, characterized in that, The vector contains the nucleic acid as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector as described in claim 3.

5. A method for preparing recombinant human type IV collagen as described in claim 1, characterized in that, Specifically, it includes the following steps: (1) Protein sequence design According to human type IV collagen, an amino acid sequence shown in SEQ ID No.1 is designed; (2) Gene design and synthesis According to the amino acid sequence of human type IV collagen described in step (1), the coding nucleic acid sequence is designed in reverse and codon-optimized to obtain the nucleotide sequence encoding recombinant human type IV collagen, and then gene synthesis is carried out to obtain a nucleic acid fragment encoding the recombinant human type IV collagen fragment; (3) Construction of expression vector: The nucleic acid fragment obtained in step (2) is ligated to the pET30a(+) plasmid through the NdeI and XhoI 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 host cell competent state by heat shock method, coated on a kanamycin-containing resistant plate, and cultured to obtain an expression strain; (5) Induced expression The expression strain obtained in step (4) is induced to express, and the bacterial liquid is collected; (6) Purification The bacterial liquid collected in step (5) is subjected to cell disruption, centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human type IV collagen.

6. The preparation method according to claim 5, characterized in that, The host cell is Escherichia coli BL21(DE3).

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