Recombinant human-like collagen polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly provides a recombinant human-like collagen polypeptide and a preparation method and application thereof, the recombinant human-like collagen polypeptide comprises an N-terminal tandem repeat sequence and a C-terminal sequence; the repetitive sequence is as shown in SEQ ID NO. 1; 1, and the C-terminal sequence is as shown in SEQ ID NO. 2. The recombinant human-like collagen polypeptide provided by the invention can correctly express active collagen with a triple helix structure in a prokaryotic expression system, so that the production cost of the active human collagen is greatly reduced, and the production efficiency is improved; compared with natural human I-type collagen, the prepared recombinant human-like collagen polypeptide has better water retention capacity and degradation resistance, and shows a cell migration promoting effect superior to that of a commercially available collagen product in a cell scratch experiment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant human-like collagen polypeptide and a preparation method and application thereof. BACKGROUND

[0002] Collagen is a macromolecular protein widely existing in mammals, accounting for about 25-35% of the total amount of proteins in the body. In the human body, collagen mainly exists in the skin, skeleton, cartilage, ligament and blood vessels, and is an extremely important structural protein in connective tissue, which plays a role in connection, support, nutrition supply and protection in connective tissue. At present, there are mainly two ways to obtain collagen: one is to extract from animal tissues such as pig bones and cow hides; the other is to obtain by using genetic engineering technology to construct an expression system.

[0003] Collagen from animals has many limitations, such as low extraction amount, risk of infection of human infectious diseases or animal source diseases, and allograft rejection. In addition, water-soluble collagen is prone to molecular chain breakage during processing, and it is difficult to completely remove the solvent during processing of water-insoluble collagen, which may cause cytotoxicity, which seriously limits the production and application of collagen.

[0004] With the development of genetic engineering technology, more and more researchers use transgenic plants and animals, insect cells, microorganisms, etc. as carriers to produce human-like collagen. The collagen obtained by using genetic engineering technology has the advantages of high safety, stable quality, no virus hidden danger, and the problems of activity, hydrophilicity and immune rejection are improved. Such collagen has excellent tissue compatibility, can be directly absorbed by the human body and participate in the construction of collagen, and is significantly effective for cell growth and wound healing, which is the development focus of modern biological medical materials.

[0005] At present, some studies have constructed recombinant human-like collagen polypeptides by repeatedly connecting the Col I alpha 1 sequence of human type I collagen as a motif. Some studies have also obtained active high expression of the polypeptide in Escherichia coli by intercepting amino acid residues at a specific position of human type III collagen, retaining the activity of collagen, and shortening the length of the polypeptide to be expressed. Some studies have introduced specific sequences, such as hydroxylated modified amino acid sequences or polypeptide fragments rich in specific amino acids, into recombinant collagen to increase the cross-linking degree of collagen and improve the stability.

[0006] However, there are still some problems in the prior art: on the one hand, it is still a challenge to correctly express active collagen with triple helix structure in prokaryotic expression system, which directly affects the production cost and efficiency; on the other hand, the stability and anti-degradation ability of recombinant collagen need to be improved, which is related to the application effect and persistence in the field of medical and cosmetic, etc. Especially for collagen products for injection, the existing technology usually needs to be injected once every 6-12 months, frequent injection not only increases the use cost, but also reduces the compliance of patients / customers.

[0007] Therefore, how to design a recombinant collagen polypeptide with good triple helix structure which can be correctly expressed in prokaryotic expression system, and has better stability and anti-degradation ability to prolong its half-life in vivo and reduce injection frequency, is a technical problem to be solved at present. SUMMARY

[0008] The purpose of the present application is to overcome the problems of high production cost, low efficiency, poor stability and anti-degradation ability of collagen obtained by using genetic engineering technology to construct an expression system in the prior art.

[0009] To this end, the present application provides a recombinant collagen polypeptide, which comprises a tandem repeat sequence at the N-terminal and a C-terminal sequence; the repeat sequence is shown as SEQ ID NO. 1; and the C-terminal sequence is shown as SEQ ID NO. 2.

[0010] Specifically, the repeat number of the above-mentioned repeat sequence is between 12-23 times.

[0011] Specifically, the repeat number of the above-mentioned repeat sequence is 16 times.

[0012] The present application also provides a gene encoding the above-mentioned recombinant collagen polypeptide, and the gene sequence is shown as SEQ ID NO. 3.

[0013] The present application also provides a recombinant expression vector carrying the above-mentioned gene sequence.

[0014] The present application also provides a recombinant engineering bacterium carrying the above-mentioned gene sequence, or containing the above-mentioned recombinant expression vector.

[0015] The present application also provides a preparation method of the above-mentioned recombinant collagen polypeptide, which comprises: expressing the above-mentioned gene to obtain a recombinant collagen polypeptide; or expressing the above-mentioned recombinant expression vector to obtain a recombinant collagen polypeptide; or fermenting and culturing the above-mentioned recombinant engineering bacterium, collecting the bacterial cells, breaking the bacteria, and separating to obtain a recombinant collagen polypeptide.

[0016] Specifically, the preparation method comprises the following steps: culturing the recombinant engineering bacteria in oscillation to an OD value of 0.6-0.8, adding an inducer to induce the culture, collecting the bacterial body, breaking the bacteria, and separating to obtain the recombinant human-like collagen polypeptide.

[0017] Specifically, the inducer comprises isopropyl-beta-D-thiogalactoside.

[0018] Specifically, the preparation method further comprises the following steps: obtaining the supernatant after the bacteria are broken, adding imidazole, filtering, and separating and purifying the recombinant human-like collagen polypeptide by using a protein purification instrument.

[0019] The application further provides application of the recombinant human-like collagen polypeptide in preparation of a collagen gel or a collagen injection.

[0020] Specifically, the collagen injection is used for preparing a cosmetic product.

[0021] Compared with the prior art, the application has the following advantages and beneficial effects:

[0022] The recombinant human-like collagen polypeptide provided by the application can correctly express active collagen with a triple helix structure in a prokaryotic expression system, greatly reduces the production cost of active human collagen and improves the production efficiency; compared with natural human type I collagen, has better water retention capacity and anti-degradation capacity, and shows better cell migration promoting effect than a commercially available collagen product in a cell scratch experiment. Experimental data show that in a 24-hour scratch healing rate test, the recombinant human-like collagen polypeptide reaches 30.27%, which is significantly higher than 18.78% of the positive control group; in a 48-hour scratch healing rate test, the product reaches 63.93%, which is also significantly higher than 38.66% of the positive control group, proving that the product has better cell repair and migration promoting capacity. In addition, the application explores the best prokaryotic expression host and fermentation conditions for expressing the recombinant human-like collagen polypeptide, obtains high expression of the recombinant human-like collagen polypeptide, and is much better than a eukaryotic expression system, and can be used for large-scale production.

[0023] The application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the triple helix structure prediction result of the recombinant human-like collagen in Example 1 of the application.

[0025] Figure 2 is the plasmid map of pET32a+ in Example 2 of the application.

[0026] Figure 3 is the PCR and enzyme digestion identification result of the recombinant plasmid in Example 2 of the application.

[0027] Figure 4 is the electrophoresis detection result of the engineered bacteria in embodiment 3 of the application.

[0028] Figure 5 is the separation and purification result of the induced expression protein of the recombinant engineered bacteria in embodiment 4 of the application.

[0029] Figure 6 is the SDS-PAGE electrophoresis detection result in embodiment 4 of the application.

[0030] Figure 7 is the high performance liquid chromatogram of the collagen freeze-dried powder in embodiment 5 of the application.

[0031] Figure 8 is the scratch healing rate test result in embodiment 6 of the application. DETAILED DESCRIPTION

[0032] The technical solutions in the application will be described clearly and completely below in conjunction with embodiments. Obviously, the described embodiments are only some of the embodiments of the application, but not all the embodiments. Although the representative embodiments of the application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the application without departing from the scope of the application. Therefore, the scope of the application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0033] The effects of the recombinant collagen-like polypeptide, the preparation method and the application thereof will be studied below through specific embodiments.

[0034] Embodiment 1

[0035] In this embodiment, the amino acid sequence of the recombinant collagen-like protein is designed, and the spatial conformation thereof is predicted.

[0036] 1. Design of the amino acid sequence of the recombinant collagen-like protein

[0037] In this embodiment, according to the periodic arrangement structural characteristics of collagen (Gly-X-Y) n, the sequence SEQ ID NO. 1 is independently set as a motif, 16 times of tandem repeats are adopted, and then the sequence shown in SEQ ID NO. 2 is connected at the C terminal to obtain the recombinant collagen-like protein.

[0038] SEQ ID NO. 1: GPKGDMGSPGPKGDRGFPGTPGIPGPLGHP;

[0039] SEQ ID NO. 2: GPPGPPGPPGPPGPP.

[0040] 2. Prediction of the spatial conformation of the recombinant collagen-like protein

[0041] The structure of the recombinant collagen-like protein designed above was predicted using online tools: https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / and AlphaFold, and the results are shown in FIG. 2, which shows that the recombinant collagen-like protein designed in this embodiment can form a good triple helix structure. Figure 1

[0042] Example 2:

[0043] In this embodiment, the recombinant expression plasmid for expressing the recombinant collagen-like protein designed in Example 1 was constructed using the plasmid pET32a (+) as the original plasmid.

[0044] 1. Design of the inserted nucleic acid molecule

[0045] The amino acid sequence of the recombinant collagen-like protein designed in Example 1 was decoded to obtain the corresponding nucleotide sequence, which was codon optimized for E. coli expression. Then, a nucleotide sequence encoding the Kpnl restriction endonuclease cleavage site and an enterokinase cleavage site were added at the 5' end, and a nucleotide sequence encoding two stop codons and a nucleotide sequence encoding the Xho I restriction endonuclease cleavage site were added at the 3' end, to facilitate cloning into the pET32a (+) expression vector. The obtained nucleic acid molecule has a total length of 1518 bp (as shown in SEQ ID NO. 3).

[0046] SEQ ID NO. 3:

[0047] ​

[0048] 2. Construction of recombinant expression plasmid

[0049] The nucleic acid molecule encoding the recombinant collagen-like protein designed in Step 1 was synthesized by chemical synthesis. The synthesized nucleic acid molecule and the plasmid pET32a (+) were subjected to enzyme digestion using Kpnl and Xhol, respectively, and then a ligation product containing the recombinant plasmid was obtained under the action of ligase. The ligation product was transformed into a DH5a E. coli cloning strain, and the recombinants were screened on LB resistant medium containing 100 μg / mL ampicillin (Amp) at 37°C. The plasmid map of pET32a (+) is shown in Figure 2 .

[0050] The recombinant plasmid obtained by culture was extracted and treated as follows:

[0051] (1) The partial recombinant plasmid was subjected to PCR amplification using a forward primer (nucleotide sequence shown in SEQ ID NO. 4) and a reverse primer (nucleotide sequence shown in SEQ ID NO. 5);

[0052] SEQ ID NO. 4: 5'-CCGACGACGATGATAAAGG-3';

[0053] SEQ ID NO. 5: 5'-GGGTTCCAGGGAAACCAC-3';

[0054] (2) The partial recombinant plasmid was subjected to double enzyme digestion using nucleases Kpnl and Xhol; and then the amplification product, the enzyme digestion product, and the recombinant plasmid without enzyme digestion were subjected to 1.0% agarose gel electrophoresis analysis. The electrophoresis result is shown in Figure 3 , in which lane 1 is the recombinant plasmid extracted from the strain, lane 2 is the product after double enzyme digestion, and lanes 3-4 are the PCR products.

[0055] As can be seen from Figure 3 , the recombinant plasmid obtained after double enzyme digestion has two bands, one of which is consistent with the size of the amplification product, with a size of 1518 bp, and the size of the target gene fragment is consistent; the other is consistent with the size of the empty original plasmid, proving that the recombinant expression plasmid for expressing the recombinant collagen-like protein is successfully constructed in this embodiment, and can be correctly amplified by PCR.

[0056] The recombinant protein with His tag is expressed with the aid of the vector start codon, and after nickel column affinity chromatography, the N-terminal tag protein and other leader amino acid sequences are cleaved by enterokinase, so that the recombinant collagen protein composed of simple collagen protein motifs can be obtained.

[0057] Example 3:

[0058] The recombinant expression plasmid constructed in Example 2 was subjected to collagen expression using a prokaryotic expression system, and the specific steps were as follows:

[0059] The recombinant plasmid obtained in Example 2 was subjected to gene sequencing, and the recombinant plasmid with correct sequencing identification was extracted and transformed into E. coli expression strain Rosetta. Then, the recombinants were screened, and the obtained recombinants were the recombinant human-like collagen with a leading sequence and fusion expression. The transformed engineering bacteria were stored in 25% glycerol and frozen in a -20°C refrigerator. The Rosetta empty bacteria without the transformed plasmid were used as a blank control, and the Rosetta bacteria containing the empty pET32a(+) plasmid were used as a negative control. The bacteria were cultured at 37°C and 220rmp until the OD value was 0.6-0.8, and then isopropyl-beta-D-thiogalactoside (IPTG) was added to a final concentration of 0.05mM. The culture was induced for 4h, and then the bacteria were collected, resuspended, broken, centrifuged, and subjected to electrophoresis detection. The results are shown in Figure 4 Lane 1 is the IPTG-induced recombinant Rosetta bacteria group, lane 2 is the non-IPTG-induced recombinant Rosetta bacteria group, lane 3 is the negative control group, and lane 4 is the Rosetta empty bacteria, i.e., the blank control group.

[0060] As can be seen from Figure 4 Compared with the blank control, the negative control, and the non-induced recombinant engineering bacteria, the induced recombinant engineering bacteria have obvious fusion protein expression, and the protein molecular weight is about 63.4kDa.

[0061] Example 4:

[0062] The collagen fusion protein successfully expressed in Example 3 was subjected to separation and purification, and the specific steps were as follows.

[0063] The fermented cells in Example 3 were collected, and after cell disruption, the supernatant was obtained, and imidazole stock solution was added to make the final concentration of imidazole in the supernatant 20 mM, and then filtered with a 0.45 μm filter membrane; using AKTA Pure chromatography system, 1 mL His Trap pre-packed column, after equilibrating the pre-packed column with 20 mM PB + 0.5 M NaCl + 20 mM imidazole, pH 7.4, the sample was loaded; after re-equilibrating with 20 mM PB + 0.5 M NaCl + 20 mM imidazole, pH 7.4 until the ultraviolet curve decreased to the baseline, the target protein was eluted with 20 mM PB + 0.5 M NaCl + 500 mM imidazole, pH 7.4, and the elution peak was collected. The eluted sample was desalted by dialysis using 50 mM Tris, pH 7.5, and filtered with a 0.22 μm filter membrane; using AKTA Pure chromatography system, 1 mL EzFast SP FF pre-packed column, after equilibrating the pre-packed column with 50 mM Tris, pH 7.5, the sample was loaded; after re-equilibrating with 50 mM Tris, pH 7.5 until the ultraviolet curve decreased to the baseline, the target protein was eluted with 50 mM Tris + 1 M NaCl, pH 7.5, and the elution peak was collected. The collected sample was detected by SDS-PAGE electrophoresis, and the results are shown in Figure 5 Figure 2. Lane 1 is the supernatant after cell disruption, lane 2 is the flow-through peak of the NI affinity chromatography, lane 3 is the elution peak of the NI affinity chromatography, lane 4 is the flow-through peak of the ion exchange SP, lane 5 is the elution peak 1 of the ion exchange, lane 6 is the elution peak 2 of the ion exchange, and lane 7 is the sample after adjusting the concentration by dialysis.

[0064] After dialysis to replace the buffer, the protein concentration was adjusted to 0.5 mg / ml, and the sample was incubated with 1 IU / mg of recombinant bovine enterokinase at 20°C for 16 h. The sample was then subjected to nickel column affinity chromatography again, and the flow-through was collected. After dialysis and concentration with PBS, the sample was detected by SDS-PAGE electrophoresis, and the results are shown in Figure 6 Figure 3. Lane 1 is the protease digestion product; lane 2 is the flow-through peak of the NI affinity chromatography, i.e., the purified recombinant collagen-like protein; and lane 3 is the elution peak of the NI affinity chromatography, which contains a band of the tag protein.

[0065] Subsequently, the endotoxin was further removed using a Detoxi-Gel endotoxin removal column.

[0066] The acid solution of collagen monomers was mixed in PBS buffer (pH 7.4). The collagen self-assembled into a high-density fibrous matrix through an instantaneous increase in pH, and then a collagen protein lyophilized powder was prepared according to the conventional lyophilization process.

[0067] Example 5:

[0068] The collagen powder obtained in Example 4 was dissolved in a suitable buffer and filtered through a 0.22 μm filter, and then the purity was identified using a high performance liquid chromatograph. The conditions of the liquid chromatograph were set as follows: mobile phase: 50 mM PB + 0.3 M Nacl, pH 6.8, flow rate: 0.3 mL / min, column temperature: 25°C, dual wavelength detection at 220 nm and 280 nm, and the chromatogram is shown in Figure 7 Figure 1, which is a single peak and has a purity of more than 95%.

[0069] Example 6

[0070] In this example, the in vitro cell repair ability of the recombinant class human collagen of Example 1 was detected by in vitro experiments, and the specific steps were as follows.

[0071] First, mark the 6-well plate with three horizontal and vertical lines in each hole using a marker pen. About (5-15) x 10 5 cells were seeded in each hole, and the goal was to reach 95-100% confluence after 24 h of culture; each group had 3 repeated holes. Three horizontal lines were drawn on the bottom of the plate using a ruler and a marker pen. After 24 h of cell culture, 10 μL of the tip was vertically aligned with the plate using a ruler, and the horizontal line was lightly pushed down to form a scratch. The cells were rinsed with PBS for 3 times to remove the scratched cells. The sample group and the positive control group were added with 2 mL of serum-free medium prepared test sample and control material, respectively, with a final concentration of 0.5 mg / mL. The blank control group was added with only serum-free medium.

[0072] The plate was cultured in a 37°C, 5% CO2 incubator, and at 0 h and 24 h, the intersection of the horizontal and vertical lines was taken as the core, and the photograph was recorded under a 40x microscope, as shown in Figure 8 Figure 2. The area of the scratch was measured, and the cell migration rate of each group was calculated by dividing the total area of the fixed scratch zone by the initial area of the fixed scratch zone. The experimental results are shown in Table 1.

[0073] Table 1: Scratch area and healing rate detection results

[0074]

[0075]

[0076]

[0077] The data in Table 1 represent the scratch area size of each group and the healing rate at different times, wherein the scratch healing area = the average value of the 0h scratch area - the corresponding scratch area, the scratch healing rate = the scratch healing area / the average value of the 0h scratch area, and the scratch area is the pixel point counted by the histogram data in the PS. According to the obtained results, it can be known that the cell culture treated by the recombinant human collagen provided by the application and the positive control (a commercially available collagen product) significantly promotes cell migration. In particular, in the experimental system considered, the recombinant human collagen provided by the application has a significantly better effect on promoting cell migration than the positive control.

[0078] In summary, the application adopts the sequence SEQ ID NO. 1 as a motif, adopts 12-23 times of tandem repeats as the N-terminal sequence, and adopts the sequence shown in SEQ ID NO. 2 derived from human collagen type III as the C-terminal sequence, to construct a new recombinant human collagen polypeptide. The recombinant human collagen polypeptide constructed by the application can be correctly expressed in a prokaryotic expression system to have an active collagen protein with a triple helix structure, greatly reducing the production cost of the active human collagen protein and improving the production efficiency. Moreover, the prepared recombinant human collagen polypeptide has better water retention capacity and anti-degradation capacity than the natural collagen protein, and can promote cell migration and growth.

[0079] The application explores the optimal expression host and fermentation conditions for expressing the above-mentioned recombinant human collagen polypeptide, and obtains high expression of the fermentation liquor, which is much better than that of the eukaryotic expression system, and can be used for large-scale production.

[0080] The above examples are only illustrative of the application and do not constitute a limitation on the protection scope of the application. Any design identical or similar to the application falls within the protection scope of the application.

Claims

1. A recombinant collagen-like polypeptide of human-like class, characterized in that: The recombinant collagen-like polypeptide comprises a tandem repeat sequence at the N-terminal and a C-terminal sequence; the repeat sequence is shown in SEQ ID NO. 1; and the C-terminal sequence is shown in SEQ ID NO.

2.

2. The recombinant collagen polypeptide of claim 1, wherein: The repeat sequence has a repeat number of 12-23.

3. A gene encoding a recombinant human collagen polypeptide as described in any one of claims 1-2, characterized in that: The gene sequence is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized by: The recombinant expression vector carries the gene sequence shown in claim 3.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineering bacteria contain the recombinant expression vector shown in claim 4.

6. A method of producing a recombinant class human collagen polypeptide, comprising, The method comprises the following steps: The recombinant engineering bacteria are fermented, and the bacteria are collected, broken, and separated to obtain the recombinant collagen-like polypeptide.

7. The method for preparing recombinant human collagen polypeptide as described in claim 6, characterized in that, The method comprises the following steps: the recombinant engineering bacteria are oscillation cultured to an OD value of 0.6-0.8, an inducing agent is added for induction culture, the bacteria are collected, broken, and separated to obtain the recombinant collagen-like polypeptide.

8. The method for preparing recombinant human collagen polypeptide as described in claim 7, characterized in that: The inducing agent comprises isopropyl-beta-D-thiogalactoside.

9. The method for preparing recombinant human collagen polypeptide as described in claim 7, characterized in that: The method further comprises the following steps: after the bacteria are broken, supernatant is obtained, imidazole is added, filtration is performed, and a protein purification instrument is used to separate and purify the recombinant collagen-like polypeptide.

10. Use of the recombinant collagen-like polypeptide shown in any one of claims 1-2 in preparation of a collagen gel or a collagen injection.

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