A recombinant human type I 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 I collagen, a preparation method thereof and an application thereof, and its amino acid sequence is shown as SEQ ID No.1. The recombinant type I collagen provided by the present invention has a high expression level, has better cell migration promotion, cell proliferation promotion and cell adhesion promotion properties, has high safety, and can be widely applied to fields such as medicine, medical devices, biological materials, tissue engineering, cosmetics and the like.

Description

Technical Field

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

[0002] Collagen is the main component of the extracellular matrix and is the most abundant and widely distributed protein in animals, accounting for about 30% of the total protein. At present, at least 28 types of collagen have been found in vertebrates. They have tissue distribution specificity and are functional proteins related to the formation, aging, and diseases of biological tissues and organs. According to whether collagen can form fibers, all collagens can be classified into two major categories: fibril-forming collagen and non-fibril-forming collagen. Among them, fibril-forming collagen includes types I, II, III, V, XI, XXIV, and XXVII collagen. Type I collagen is the most commonly existing collagen, accounting for about 90% of the total collagen in animals and nearly 20% of the total protein mass. Human type I collagen is a heterotrimer [αl(I)]2α2(I) composed of two different α-chains, αl(I) and α2(I).

[0003] Type I collagen has excellent hemostatic and tissue repair capabilities and has been widely used in medical, cosmetic, and other fields. Type I collagen is mainly obtained by animal extraction. However, there are problems such as poor batch-to-batch consistency, poor water solubility, and the risk of virus transmission in animal-extracted collagen. Obtaining recombinant collagen by recombinant expression can well solve the above problems. The recombinant expression of collagen generally adopts several methods such as full-length expression, fragment expression, and small-fragment repetition. Full-length expression is relatively difficult, generally with a very low expression level and difficult to achieve large-scale production; while fragment and small-fragment repetition are relatively easy to express, and high-expression strains can be screened to achieve industrialization. However, not any random fragment can be expressed. Most sequences have a very low expression level or do not express at all. Therefore, the design of the recombinant collagen sequence is the key to the preparation of recombinant collagen.

[0004] In view of this, it is of great significance to find a recombinant type I collagen sequence that can be highly expressed and has excellent biological properties. Summary of the Invention

[0005] Through different sequence design and screening work, the present invention provides a recombinant human type I collagen with high expression and good cell activity, and simultaneously provides a preparation method and an application thereof.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] On the one hand, the present invention provides a recombinant human type I collagen, and the recombinant collagen comprises one or more of the following characteristics:

[0008] (1) Its amino acid sequence is as shown in SEQ ID No.1, specifically as follows:

[0009] MHHHHHHGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGP;

[0010] (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;

[0011] (3) An amino acid sequence having more than 80% identity with the amino acid sequence shown in SEQ ID No.1.

[0012] On the other hand, the present invention provides a vector, and the vector contains a nucleic acid encoding the above-mentioned collagen.

[0013] On the other hand, the present invention provides a host cell, and the host cell contains the above-mentioned vector.

[0014] On the other hand, the present invention provides a method for preparing the above-mentioned recombinant human type I collagen, specifically comprising the following steps:

[0015] (1) Protein sequence design

[0016] According to human type I collagen, an amino acid sequence as shown in SEQ No.1 is designed;

[0017] (2) Gene design and synthesis

[0018] Reverse-design the coding nucleic acid sequence according to the amino acid sequence of recombinant human type I collagen described in step (1), and perform codon optimization to obtain the nucleotide sequence encoding recombinant human type I collagen, and then perform gene synthesis to obtain the nucleic acid fragment encoding the recombinant human type I collagen fragment;

[0019] (3)Construction of expression vector:

[0020] Connect it to the pET30a(+) plasmid through the NdeI and XhoI multiple cloning sites to obtain the expression plasmid;

[0021] (4)Construction and screening of expression strains:

[0022] Transfer the plasmid described in step (3) into the host cell competent state by heat shock method, coat it on the resistance plate containing kanamycin, and culture it to obtain the expression strain;

[0023] (5)Induced expression

[0024] Induce the expression strain obtained in step (4) to express, and collect the bacterial liquid;

[0025] (6)Purification

[0026] Perform cell disruption, centrifugation, affinity chromatography, ion exchange chromatography, desalting, and lyophilization on the bacterial liquid collected in step (5) to obtain the lyophilized product of recombinant human type I collagen.

[0027] As some preferred embodiments of the present invention, the host cell is Escherichia coli BL21(DE3).

[0028] On the other hand, the present invention provides a use of the above-mentioned recombinant human type I collagen in the field of medical devices.

[0029] The beneficial effects produced by adopting the above technical solutions are as follows:

[0030] (1)The recombinant human type I collagen provided by the present invention has [A1] to form a gel.

[0031] (2)The recombinant human type I collagen provided by the present invention has been experimentally verified to have better cell migration promotion, cell proliferation promotion, and cell adhesion promotion properties.

[0032] (3)The preparation method of the recombinant human type I collagen of the present invention has a high yield and is suitable for large-scale industrial production. Description of the drawings

[0033] 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.

[0034] Figure 1 Electrophoresis diagram of the purified recombinant type I collagen sample;

[0035] Figure 2 It is the result diagram of the screening of HC37 shake flasks;

[0036] Figure 3 It is the effect diagram of promoting cell migration of recombinant type I collagen at different concentrations;

[0037] Figure 4 It is the effect diagram of promoting cell proliferation of recombinant type I collagen at different concentrations;

[0038] Figure 5 It is the effect diagram of promoting cell adhesion of recombinant type I collagen at different concentrations;

[0039] Figure 6 It is the stability result of the freeze-dried product of recombinant type I collagen;

[0040] Figure 7 It is the thermosensitive property of recombinant type I collagen. Detailed implementation mode

[0041] To make the objectives, technical solutions and advantages of the present invention clearer, the following describes the invention clearly and completely in combination with specific embodiments.

[0042] Example 1 Gene design and synthesis

[0043] (1) Gene design:

[0044] According to the sequence characteristics of human type I collagen, the collagen sequence HC90 was designed. Its amino acid sequence is shown in SEQ ID NO.1, which is a tandem repeat of amino acids 671-750 of the α1 chain, and the number of repeats is 4. Its N-terminus starts with methionine to reduce the protein degradation rate; immediately followed by a His-Tag of 6 histidines for easy identification and purification. The amino acid sequence of the recombinant human type I collagen fragment is as follows:

[0045] SEQ ID NO.1

[0046] MHHHHHHGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGP。

[0047] The coding nucleic acid sequence was reverse-designed using the online design tool Jcat (http: / / www.jcat.de / ), 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 I collagen was obtained.

[0048] (2)Gene synthesis:

[0049] According to the nucleic acid sequence, GenScript Biotech Corporation synthesized the nucleic acid fragment encoding the recombinant human type I collagen fragment.

[0050] Example 2 Construction of expression vector pET30a(+)-HC90

[0051] The nucleic acid fragment obtained in Example 1 was ligated to the pET30a(+) plasmid through the multiple cloning sites NdeI and XhoI to obtain the recombinant plasmid pET30a(+)-HC90.

[0052] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-HC90

[0053] 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 the residual liquid to flow out as much 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 the residual liquid to flow out as much 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(+)-HC90 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 no shaking, and immediately cool it on ice; Add 500 μl of liquid LB medium, mix well, and place it in a 37°C shaker and shake at low speed 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 an antibiotic (kanamycin), and incubate it inverted in a 37°C incubator. The grown colonies are the expression strain BL21(DE3) / pET30a(+)-HC90.

[0054] Example 4 Induced Expression of the Expression Strain BL21(DE3) / pET30a(+)-HC90

[0055] Pick a single colony of the expression strain BL21(DE3) / pET30a(+)-HC90 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.

[0056] Example 5 Purification of Recombinant Human Type I Collagen HC90

[0057] The specific purification steps of HC90 are as follows:

[0058] 1. Bacterial cell disruption

[0059] Resuspend the bacterial cells prepared in Example 4 with an appropriate amount of cell disruption solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0). The ratio of bacterial cells to cell disruption solution (m / v) is 1:5 - 1:20, and 1:10 is selected in this example. Disrupt the bacterial cells with a high-pressure homogenizer. Centrifuge the disrupted cell suspension at 12,000 g for 1 h, collect the supernatant, and filter the supernatant through a 0.45 μm filter membrane.

[0060] 2. Affinity chromatography

[0061] (1) Column equilibration:

[0062] Equilibrate the Ni Sepharose FF chromatography column with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0) until the baseline is stable.

[0063] (2) Sample loading:

[0064] Load the supernatant onto the chromatography column and control the column retention time to be no less than 5 min.

[0065] (3) Column washing:

[0066] Wash the chromatography column with buffer A until the baseline is stable, with a volume of no less than 5 column volumes (CV).

[0067] (4) Elution of the target protein:

[0068] Elute with 80% Buffer A and 20% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole), collect the eluate into a new centrifuge tube to obtain a solution containing recombinant human type I collagen HC90.

[0069] 3. Ion exchange chromatography

[0070] (1) Sample treatment:

[0071] Dilute the affinity chromatography eluate with ion exchange solution A (20 mM Tris, pH 8.0) or ultrafiltrate and replace it until the conductivity is less than 5 mS / cm.

[0072] (2) Column equilibration:

[0073] Equilibrate the SP Sepharose FF chromatography column with buffer A until the baseline is stable.

[0074] (3) Sample loading:

[0075] Load the renaturation solution onto the chromatography column and control the column retention time to be no less than 5 min.

[0076] (4)Column washing:

[0077] Wash the chromatography column with buffer A until the baseline is stable, not less than 5 CVs.

[0078] (5)Elution of target protein:

[0079] Elute with 75% Buffer A and 25% Buffer B (20 mM Tris, 1 M NaCl, pH 8.0), collect the eluate into a new centrifuge tube to obtain a solution containing recombinant human type I collagen HC90.

[0080] 4. Desalting

[0081] Replace the above solution with purified water using a 5 kDa ultrafiltration membrane device by equal-volume replacement to remove the salt in the solution, filter it through a 0.22 μm filter membrane and then lyophilize to obtain the recombinant collagen lyophilized product, with a yield of 9 g / L fermentation broth.

[0082] Example 6 SDS-PAGE protein electrophoresis detection

[0083] Sample treatment: Collect the bacterial cells prepared in Example 4 and the purified samples in Example 5, add loading buffer and mix well, heat in a boiling water bath for 10 min, and cool naturally for standby. Use the GenScript SurePAGE™ precast gel (4-12%) for loading and electrophorese at 140 V for 45-55 minutes until the bromophenol blue band runs to the bottom of the gel.

[0084] 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 40% ethanol and 10% acetic acid solution. 2) Prepare the destaining 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 take out the gel, and then put it into a staining container containing 100 ml of the staining solution. 4) Cover the container lid and put it into the microwave oven and heat at high heat for 8 minutes. To avoid danger, please note that the solution should not be allowed to boil. 5) Take out the staining container from the microwave oven, place it on a shaker and gently shake 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 destaining solution. 8) Cover the lid, put it into the microwave oven and heat at high heat for 8 minutes. 9) Pour out the destaining solution, add fresh destaining solution, and repeat step 8. 10) Take out from the microwave oven, place it on a shaker and gently shake at room temperature until the background is clear.

[0085] As Figure 1 shown, after two-step chromatography of recombinant type I collagen, the protein purity is already very high. For the desalted sample, almost no band of impurity protein can be seen in electrophoresis.

[0086] Example 7 Design and Screening of Other Recombinant Type I Collagen Sequences

[0087] Referring to Example 1, other different collagen sequences were designed. The amino acid sequences and corresponding nucleotide sequences are shown in Table 1:

[0088] Table 1 List of Other Recombinant Protein Sequences

[0089]

[0090] HC37, HC38, HC89, HC91, HC106 and HC107 were constructed according to the methods of Examples 2 to 4. Single colonies were picked and verified by shake flask induction, but no expression was observed. Figure 2 Figure 25 is the SDS-PAGE detection result of the shake flask induction sample of HC37. There is no obvious accumulation of the target protein near 25 kDa. The screening results of other sequences are similar and not all listed.

[0091] Effect Example 1 Recombinant Human Type I Collagen HC90 Promotes Cell Migration Assay

[0092] Use a marker pen to make marks by drawing three equal parts horizontally and vertically on the back of each well of a 6-well culture plate. Seed about 10×10 5 L929 mouse fibroblasts in each well. When the cells reach 95% confluence, use a 10 µL pipette tip to scratch the cells to form a cell scratch. Rinse the cells 3 times with PBS to remove the scratched cells. Take a photo under the microscope with the intersection of the horizontal and vertical scratches as the center, and record the scratch area at this time, which is recorded as the scratch area at 0 h. Add culture medium to the blank control group and add HC90 solution to the sample group, and continue to culture. Take photos under the microscope after culturing for 24 h and 48 h.

[0093] As Figure 3 shown, recombinant human type I collagen HC90 has good performance in promoting cell migration.

[0094] Effect Example 2 Recombinant Human Type I Collagen HC90 Promotes Cell Proliferation Assay

[0095] After the HaCaT cells were digested with trypsin, they were seeded at 8×10 3Cells were inoculated into the wells. After inoculating the cells into the 96-well culture plate, it was placed in an incubator at 37 °C and 5% CO2 for 24 h. 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 HC90 solution was added to each well. The 96-well culture plate was placed in an incubator at 37 °C and 5% CO2 for 24 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 I collagen HC90 has a good cell proliferation effect.

[0096] Effect Example 4: Recombinant human type I collagen HC90 promotes cell adhesion test

[0097] Mouse fibroblast NIH / 3T3 cells were digested and counted, and 1×10 5 cells were inoculated into each well of a 96-well plate at 100 μL / well. The blank control group (BC) was only added with PBS, the sample group (added with 2, 4, 6, 8, 10 mg / ml HC90), and the positive control group (the sample maximum enzyme activity control group, PC). The culture medium was discarded, and the samples were each washed once with PBS buffer and treated for 24 h (5% CO2, 37 °C, >90% humidity). One hour before the scheduled detection time, the "sample maximum enzyme activity control well" was treated with LDH release reagent. The addition amount was 10% of the original culture medium volume. After adding the LDH release reagent, it was pipetted and mixed repeatedly, and incubation was continued; at the scheduled time, the cell culture plate was centrifuged at 400 g for 5 min using a multi-well centrifuge. 120 μL of the supernatant was taken into a new 96-well plate; 60 μL of LDH detection solution was added to each well, mixed well, and incubated in the dark at room temperature for 30 min; the absorbance was measured at 490 nm. The results are as Figure 5 shown. Compared with the control group, HC90 at a concentration of 6 mg / ml - 10 mg / ml can increase the level of LDH secreted by mouse fibroblast NIH / 3T3 cells, thereby improving the cell adhesion ability.

[0098] Effect Example 5: Stability study of recombinant human type I collagen HC90

[0099] The freeze-dried product of HC90 was taken and placed under the conditions of 25 °C ± 2 °C and relative humidity of 55% - 65%. Samples were taken at 1 month, 3 months, 6 months, 12 months, and 24 months for SDS-PAGE protein electrophoresis detection. As Figure 6 shown, there was no obvious degradation of HC90 after storage for 24 months.

[0100] As can be seen from the above embodiments, not any fragment of recombinant type I collagen can achieve high expression. The recombinant human type I collagen of the present invention has a high expression level; and it can promote cell proliferation, migration and adhesion. At the same time, it has good stability, which is beneficial to the development of pharmaceutical products.

[0101] Effect Example 6 Gel-forming property of recombinant type I collagen HC90

[0102] Prepare a 10 mg / ml solution of HC90 and place it in 2 EP tubes, 1 ml per tube. One tube is stored at room temperature and the other is stored at 4°C. After leaving it overnight, observe the state of the solution. As Figure 7 shown, the one stored at room temperature remains in a solution state, while the one stored at 4°C turns into a gel state, without fluidity, and will not fall or flow when inverted or shaken. This shows that HC90 has thermosensitivity and can form a gel at low temperature.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on 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 water-soluble recombinant human type I collagen, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. A carrier, characterized in that, The vector contains the nucleic acid encoding the collagen as claimed in claim 1.

3. A host cell, characterized in that, The host cell contains the vector as claimed in claim 2.

4. A preparation method of the water-soluble recombinant human type I collagen as described in claim 1, characterized in that, Specifically, it includes the following steps: (1) Protein sequence design According to human type I collagen, an amino acid sequence as shown in SEQ ID No.1 is designed; (2) Gene design and synthesis According to the amino acid sequence of human type I collagen described in step (1), the encoding nucleic acid sequence is designed in reverse and codon-optimized to obtain the nucleotide sequence encoding recombinant human type I collagen, and then gene synthesis is carried out to obtain the nucleic acid fragment encoding the recombinant human type I collagen; (3) Construction of expression vector: It is ligated to the pET30a(+) plasmid through the NdeI and XhoI multiple cloning sites to obtain the expression plasmid; (4) Construction and screening of expression strain: The plasmid described in step (3) is transferred into the host cell competent state by heat shock method, coated on the resistance plate containing kanamycin, and cultured to obtain the 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 the purification of recombinant human type I collagen.

5. The preparation method according to claim 4, characterized in that, The host cell is Escherichia coli BL21(DE3).

6. Use of a water-soluble recombinant human type I collagen as claimed in claim 1 in the preparation of tissue repair products.

Images