Recombinant human I-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 I-type collagen as well as a preparation method and application thereof, and the amino acid sequence of the recombinant human I-type collagen is shown as SEQ ID No.1. The recombinant I-type collagen provided by the invention is relatively high in expression quantity, has relatively good cell migration promoting, cell proliferation promoting and cell adhesion promoting properties, is high in safety, and can be widely applied to the fields of medicines, medical instruments, biological materials, tissue engineering, cosmetics and the like.

Description

Technical Field

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

[0002] Collagen is the main component of the extracellular matrix and 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 disease of tissues and organs of organisms. According to whether collagen can form fibers, all collagens can be divided into two categories: fibril-forming collagen and non-fibril-forming collagen. The fibril-forming collagens include type I, II, III, V, XI, XXIV and XXVII collagens. Type I collagen is the most common collagen, accounting for about 90% of the total collagen in animals and nearly 20% of the total protein. 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 hemostasis and tissue repair capabilities and has been widely used in medical and beauty fields. Type I collagen is mainly obtained by animal extraction. However, animal-extracted collagen has problems such as poor batch consistency, poor water solubility, and the risk of virus transmission. Obtaining recombinant collagen through recombinant expression can solve the above problems well. The recombinant expression of collagen generally adopts several methods such as full-length expression, fragment expression, and small fragment repetition. Full-length expression is more difficult, and the expression level is generally very low, making it difficult to achieve large-scale production; while fragments and small fragment repetitions are easier to express, and high-expression strains can be screened to achieve industrialization. However, not any random fragment can be expressed, and most sequences have very low expression levels, or are not expressed at all. Therefore, the design of recombinant collagen sequence is the key to the preparation of recombinant collagen.

[0004] In view of this, it will be 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] The present invention provides a recombinant human type I collagen with high expression and good cell activity through different sequence designs and screenings, and also provides a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is: In one aspect, the present invention provides a recombinant human type I collagen, wherein the recombinant collagen comprises one or more of the following characteristics: (1) Its amino acid sequence is shown in SEQ ID No. 1, which is as follows: MHHHHHHGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGD AGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGP; (2) Its amino acid sequence is modified by substitution, deletion or addition of one or more amino acids based on SEQ ID No.1; (3) An amino acid sequence that is more than 80% identical to the amino acid sequence shown in SEQ ID No: 1.

[0007] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned recombinant human type I collagen, wherein the nucleotide sequence of the recombinant human type I collagen comprises one or more of the following features: (1) The nucleotide sequence is shown in SEQ ID No. 2, which is as follows: .

[0008] (2) Any nucleotide sequence obtained by replacing synonymous codons in (1).

[0009] In yet another aspect, the present invention provides a vector comprising the nucleotide sequence shown in SEQ ID No.2.

[0010] In still another aspect, the present invention provides a host cell, wherein the host cell contains the above-mentioned vector.

[0011] In another aspect, the present invention provides a method for preparing the above-mentioned recombinant human type I collagen, which specifically comprises the following steps: (1) Protein sequence design Based on human type I collagen, an amino acid sequence as shown in SEQ No. 1 was designed; (2) Gene design and synthesis Reversely designing a nucleic acid sequence encoding the recombinant human type I collagen according to the amino acid sequence of the recombinant human type I collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type I collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type I collagen fragment; (3) Construction of expression vector: The expression plasmid was obtained by ligating the plasmid to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites; (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, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Inducing the expression strain obtained in step (4) to express, and collecting 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 recombinant human type I collagen freeze-dried product.

[0012] As some preferred embodiments of the present invention, the host cell is Escherichia coli BL21 (DE3).

[0013] In another aspect, the present invention provides a use of the above-mentioned recombinant human type I collagen in the field of medical devices.

[0014] The beneficial effects of adopting the above technical solution are: (1) The recombinant human type I collagen provided by the present invention has [A1] gel formation.

[0015] (2) The recombinant human type I collagen provided by the present invention has been experimentally verified to have better performance in promoting cell migration, cell proliferation and cell adhesion.

[0016] (3) The method for preparing recombinant human type I collagen of the present invention has high yield and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the specific implementation manner of the present invention, the drawings required for describing the specific implementation manner are briefly introduced below.

[0018] Figure 1 This is the electrophoresis diagram of the purified sample of recombinant type I collagen; Figure 2 This is the result of HC37 shake flask screening; Figure 3 The figure shows the effect of different concentrations of recombinant type I collagen on promoting cell migration; Figure 4 This is a diagram showing the cell proliferation effects of different concentrations of recombinant type I collagen; Figure 5 The figure shows the cell adhesion promoting effect of recombinant type I collagen at different concentrations; Figure 6 This is the stability result of the lyophilized recombinant type I collagen; Figure 7 Thermosensitive properties of recombinant type I collagen. DETAILED DESCRIPTION

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

[0020] Example 1 Gene Design and Synthesis (1) Genetic design: 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, with a repeat count of 4. Its N-terminus starts with methionine to reduce the degradation rate of the protein; 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 shown below: SEQ ID NO.1 MHHHHHHGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGD AGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGPKGDRGDAGP.

[0021] The coding nucleic acid sequence was reversely designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for host E. coli expression. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human type I collagen was obtained, and the nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2: ATGGGGGTAAAAGGAGAAGCTGGTCTACCCGGAACCCCGGGTCCAACGGGTCCGGGCAGGTCAGAAAGGCGAACCGGGTTCTGATGGTATCCCGGGCAGCGCAGGCGAAAAGGGCGAGCCGGGCCTCCCGGGACGCGGTTT CCCGGGTTTCCCGGGCGCGAAAGGCGACAAAGGCAGCAAGGGCGAGGTGGGCTTCGACGGTCGTAATGGTGAGAAGGGTGAAACCGGTGCGCCTGGTCTGAAAGGCGAGAATGGCTTGCCGGGCGAGAACGGCGCGCCGG GCCCGATGGGTCCGCGTGGTGCGCCAGGTGAGCGCGGTAGACCGGGCCTTCCGGGCGCTGCCGGCGCCGTGGCGGGGCGGGCTCCCTGGGTGCCGGTGGCGCGTTTGGTGAAGCAGCGGGTGACCGTGGTCCGTACGGC ACCGATATTGGTCCGGGCGGTGGTTATGGTGCCGCTGCTGAAGGTGGTATGTACGCGGGAAACGGCGGTTTGCTGGGTGCGGATTTTGCTGGCGACCTGGATTATAACGAACTGGCACATCATCACCACCACCATCACTAA (2) Gene synthesis: Based on the nucleic acid sequence shown above, GenScript Biotech Corporation synthesized a nucleic acid fragment encoding a recombinant human type I collagen fragment.

[0022] Example 2 Construction of expression vector pET30a(+)-HC90 The nucleic acid fragment SEQ ID NO.2 obtained in Example 1 was ligated to the pET30a(+) plasmid via the multiple cloning sites NdeI and XhoI to obtain the recombinant plasmid pET30a(+)-HC90.

[0023] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-HC90 The construction of the expression strain refers to the method described in the Molecular Cloning Experiment Guide (3rd Edition) (J. Sambrook et al.), and 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 at 37°C overnight with shaking; Add 0.5 ml of overnight culture medium to the LB flask, and culture with vigorous shaking at 37°C for about 2 hours to allow the bacteria to grow to the early logarithmic stage; transfer the bacteria to a 50 ml polypropylene tube pre-cooled with ice under sterile conditions and place on ice for 10 minutes; centrifuge at 4°C, 4000 rpm, pour out 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 precipitate, and place on ice for 30 minutes; centrifuge at 4°C, 3000 rpm, pour out 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 precipitate (if you want to prepare competent cells for storage at -70°C, add 0.1 mol / L CaCl2 containing 20% ​​glycerol CaCl2 suspended bacteria), placed at 4 ° C for 5 to 24 hours, aspirate 200 μl of competent cell suspension, add the recombinant plasmid pET30a (+) -HC90 prepared in Example 2 (volume <10 μl, DNA <50 ng), gently mix, and place on ice for 30 minutes; heat shock in a 42 ° C water bath for 90 seconds, and immediately cool on ice; add 500 μl of liquid LB culture medium, mix, and place in a 37 ° C shaker for low speed shaking for 45 minutes (it can also be directly placed in a 37 ° C water bath for 1 hour after adding LB to suspend the cells); aspirate the transformed cells and spread them on a plate with antibiotics (kanamycin), place them in a 37 ° C incubator and invert them for culture, and the colonies grown are the expression strain BL21 (DE3) / pET30a (+) -HC90.

[0024] Example 4 Induced expression of expression strain BL21(DE3) / pET30a(+)-HC90 A single colony of the expression strain BL21(DE3) / pET30a(+)-HC90 prepared in Example 3 was selected and placed in LB liquid medium containing 50 μg / mL Kan. After culturing overnight at 37°C and 200 r / min, it became an activated seed. Then, it was inoculated into a 5L fermenter containing 3L CM medium at a 3% inoculum. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. When OD 600 When the expression reached 60, IPTG (isopropyl-β-D-thiogalactoside) 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.

[0025] Example 5 Purification of recombinant human type I collagen HC90 The specific purification steps of HC90 are as follows: 1. Breaking bacteria 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 8.0), with the ratio (m / v) of bacterial cells to lysis solution being 1:5-1:20, and 1:10 was selected in this example. The bacterial cells were ruptured using a high-pressure homogenizer. The ruptured bacterial solution was centrifuged at 12000 g for 1 h, and the supernatant was collected and filtered using a 0.45 μm filter membrane.

[0026] 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 8.0) until the baseline was stable.

[0027] (2) Loading: The supernatant was added to the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0028] (3) Column flushing: Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0029] (4) Target protein elution: The solution was eluted with 80% Buffer A and 20% 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 I collagen HC90.

[0030] 3. Ion exchange chromatography (1) Sample processing: The affinity chromatography eluate was diluted with ion exchange solution A (20 mM Tris, pH 8.0) or ultrafiltered to a conductivity of less than 5 mS / cm.

[0031] (2) Column balance: The SP Sepharose FF column was equilibrated with buffer A until the baseline was stable.

[0032] (3) Loading: The refolding solution was added to the chromatography column, and the column retention time was controlled to be no less than 5 minutes.

[0033] (4) Column flushing: Rinse the column with buffer A until the baseline is stable and not less than 5 CV.

[0034] (5) Target protein elution: The solution was eluted with 75% Buffer A and 25% Buffer B (20 mM Tris, 1 M NaCl, pH 8.0), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type I collagen HC90.

[0035] 4. Desalination The above solution was replaced with purified water by equal volume replacement using a 5 kDa ultrafiltration membrane device to remove the salt in the solution, and then filtered with a 0.22 μm filter membrane and freeze-dried to obtain a recombinant collagen freeze-dried product with a yield of 9 g / L fermentation liquid.

[0036] Example 6 SDS-PAGE protein electrophoresis detection Sample treatment: Collect the bacteria prepared in Example 4 and the purified samples in Example 5, add loading buffer and mix evenly, boil in a boiling water bath for 10 min, cool naturally and set aside. Select GenScript SurePAGE™ precast gel (4-12%) for sample loading, and perform electrophoresis at 140 V for 45-55 min until the bromophenol blue band runs to the bottom of the gel.

[0037] Coomassie Brilliant Blue R-250 Staining using a microwave oven: 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 solution. 2) Prepare the destaining solution: Dissolve 10% (V / V) ethanol and 7.5% (V / V) acetic acid together. 3) After electrophoresis, pry open the gel sheet to remove the gel and place it in a staining container with 100 ml of staining solution. 4) Cover the container and place it in a microwave oven on high heat for 8 minutes. To avoid danger, be careful not to let the solution boil. 5) Remove the staining container from the microwave and place it on a destaining shaker at room temperature for 5 minutes. 6) Pour off the staining solution and carefully wash the gel with deionized water. 7) Pour off the deionized water and add 100 ml of destaining solution. 8) Cover the container and place it in a microwave oven on high heat for 8 minutes. 9) Pour out the destaining solution, add new destaining solution, and repeat step 8. 10) Take it out from the microwave, place it on a destaining shaker at room temperature and shake gently until the background is clear.

[0038] like Figure 1 As shown, after two steps of chromatography, the purity of recombinant type I collagen is already very high, and in the desalted sample, almost no impurity protein bands can be seen in the electrophoresis.

[0039] Example 7 Design and screening of other recombinant type I collagen sequences Referring to Example 1, other different collagen sequences were designed, and the amino acid sequences and corresponding nucleotide sequences are shown in Table 1: Table 1 Other recombinant protein sequences HC37, HC38, HC89, HC91, HC106 and HC107 were constructed according to the methods of Examples 2 to 4, and single colonies were picked and verified by shake flask induction, and no expression was found. Figure 2 The results of SDS-PAGE detection of shake flask induced samples of HC37 show that there is no obvious accumulation of target protein around 25kda. The screening results of other sequences are similar and are not listed in full.

[0040] Effect Example 1 Recombinant human type I collagen HC90 promotes cell migration test Mark each well in the 6-well culture plate with a marker pen and mark it in three equal parts horizontally and vertically. 5 L929 mouse fibroblasts. When the cells reached 95% confluence, 10 µL pipette tip was used to scratch the cells. The cells were rinsed 3 times with PBS to remove the scratched cells. The intersection of the horizontal and vertical lines was taken as the core, and the scratch area at this time was recorded and recorded as the scratch area at 0 h. The blank control group was added with culture medium, and the sample group was added with HC90 solution and cultured continuously. After 24 h and 48 h of culture, photos were taken under a microscope.

[0041] like Figure 3 The results showed that recombinant human type I collagen HC90 has a good ability to promote cell migration.

[0042] Effect Example 2 Recombinant human type I collagen HC90 promotes cell proliferation test HaCaT cells were digested with trypsin and cultured at 8 × 10 3 / well for cell inoculation. After the 96-well culture plate was inoculated with cells, it was placed in a 37 ℃, 5% CO2 incubator for 24 hours. The culture medium in the wells was discarded, 100 μL of culture medium was added to each well of the blank control group, and 100 μL of HC90 solution was added to each well of the sample group. The 96-well culture plate was placed in a 37 ℃, 5% CO2 incubator for 24 hours. 10 μL of CCK8 reagent was added to each well and placed in the incubator for incubation for 2 hours. After incubation, the absorbance value of each well at a wavelength of 450 nm was detected using an enzyme-labeled instrument. Figure 4 As shown, recombinant human type I collagen HC90 has a good cell proliferation effect.

[0043] Effect Example 4 Recombinant human type I collagen HC90 promotes cell adhesion test Mouse fibroblasts NIH / 3T3 were digested and counted, and 1×10 cells were seeded per well in a 96-well plate. 5cells, 100 μL / well. The blank control group (BC) only added PBS, the sample group (added 2, 4, 6, 8, 10 mg / ml HC90), and the positive control group (sample maximum enzyme activity control group, PC). The culture medium was discarded, and the samples were washed once with PBS buffer and treated for 24 h (5% CO2, 37 ℃, >90% humidity). One hour before the scheduled detection time, the "sample maximum enzyme activity control well" was treated with LDH release reagent. The amount added was 10% of the original culture medium volume. After adding the LDH release reagent, repeatedly blow and mix, and continue incubation; when the scheduled time comes, the cell culture plate is centrifuged at 400 g in a multi-well centrifuge for 5 min. Take 120 μL of supernatant in a new 96-well plate; add 60 μL of LDH detection solution to each well, mix, and incubate at room temperature in the dark for 30 min; the absorbance value is measured at 490 nm. The results are as follows Figure 5 As shown, compared with the control group, HC90 at a concentration of 6mg / ml-10mg / ml can increase the level of LDH secreted by mouse fibroblast NIH / 3T3 cells, thereby improving the ability of cell adhesion.

[0044] Effect Example 5: Stability Study of Recombinant Human Type I Collagen HC90 Take the HC90 freeze-dried product and place it at 25℃±2℃ and relative humidity of 55%-65%. Take samples for SDS-PAGE protein electrophoresis at 1 month, 3 months, 6 months, 12 months and 24 months. Figure 6 As shown in the figure, HC90 showed no obvious degradation after 24 months of storage.

[0045] It can be seen from the above examples that not all recombinant type I collagen fragments can achieve high expression, and the recombinant human type I collagen of the present invention has high expression and can promote cell proliferation, migration and adhesion. At the same time, it has good stability, which is conducive to the development of preparation products.

[0046] Effect Example 6: Gelation performance of recombinant type I collagen HC90 Prepare a 10mg / ml solution of HC90 and put it into two EP tubes, 1ml / tube. Store one tube at room temperature and the other at 4℃. After leaving overnight, observe the state of the solution. Figure 7 As shown in the figure, the solution stored at room temperature is still in solution state, while the solution stored at 4°C becomes gel state, without fluidity, and will not fall or flow when placed upside down or shaken. This indicates that HC90 is temperature sensitive and can form gel at low temperatures.

[0047] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 nucleic acid encoding the recombinant human type I collagen according to claim 1, characterized in that: The nucleotide sequence of the recombinant human type I collagen is shown in SEQ ID No.

2.

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

4. A host cell, characterized in that The host cell contains the vector according to claim 3.

5. A method for preparing the water-soluble recombinant human type I collagen as claimed in claim 1, characterized in that: The specific steps include: (1) Protein sequence design Based on human type I collagen, an amino acid sequence as shown in SEQ No. 1 was designed; (2) Gene design and synthesis Reversely designing a nucleic acid sequence encoding the recombinant human type I collagen according to the amino acid sequence of the recombinant human type I collagen described in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human type I collagen, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type I collagen fragment; (3) Construction of expression vector: The expression plasmid was obtained by ligating the plasmid to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites; (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, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Inducing the expression strain obtained in step (4) to express, and collecting the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption, inclusion body washing, denaturation, and renaturation, and recombinant human type I collagen is purified using a chromatography column.

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

7. Use of the water-soluble recombinant human type I collagen as claimed in claim 1 in the preparation of pharmaceutical products, medical devices, biomaterials, tissue engineering and cosmetics.

Images