A recombinant collagen-like polypeptide and preparation method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and specifically provides a recombinant human-like collagen polypeptide, a preparation method and application thereof. The recombinant human-like collagen polypeptide comprises a tandem repeat sequence at an 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. The recombinant human-like collagen polypeptide provided in the application can correctly express active collagen with a triple helix structure in a prokaryotic expression system, greatly reducing the production cost of active human collagen and improving the production efficiency. The prepared recombinant human-like collagen polypeptide has better water retention capacity and anti-degradation capacity than natural human type I collagen, and shows a cell migration promoting effect superior to that of a commercial 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 body, 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 recombinant human collagen

[0041] The recombinant human collagen protein designed above was structurally predicted using online tools: https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / and AlphaFold. The results are as follows: Figure 1 As shown, the recombinant human collagen designed in this embodiment can form a good triple helix structure.

[0042] Example 2:

[0043] This embodiment aims to express the recombinant human collagen designed in Example 1. A recombinant expression plasmid for expressing the recombinant human collagen was constructed using plasmid pET32a(+) as the original plasmid.

[0044] 1. Insertion of nucleic acid molecules design

[0045] The amino acid sequence of the recombinant human collagen designed in Example 1 was decoded to obtain the corresponding nucleotide sequence, and the expression codons in *E. coli* were optimized. Then, nucleotide sequences encoding the KpnI restriction endonuclease cleavage site and the enterokinase cleavage site were added to the 5' end, and nucleotide sequences encoding two stop codons and the XhoI restriction endonuclease cleavage site were added to the 3' end to facilitate cloning into the pET32a(+) expression vector. The resulting nucleic acid molecule was 1518 bp in length (as shown in SEQ ID NO.3).

[0046] SEQ ID NO.3:

[0047]

[0048] 2. Construction of recombinant expression plasmids

[0049] The nucleic acid molecule encoding recombinant human collagen designed in step 1 was synthesized using a chemical synthesis method. The synthesized nucleic acid molecule and plasmid pET32a(+) were digested with the restriction enzymes KpnI and XhoI, respectively. Then, a ligation product containing the recombinant plasmid was obtained using a ligase. This ligation product was transformed into a DH5α *E. coli* clone strain and cultured at 37°C on LB resistant medium supplemented with 100 µg / mL ampicillin (Amp). Recombinants were screened. The plasmid map of pET32a+ is shown below. Figure 2 As shown.

[0050] The recombinant plasmid obtained from the culture was extracted and processed as follows:

[0051] (1) PCR amplification of some recombinant plasmids was performed using forward primers (nucleotide sequences as shown in SEQ ID NO.4) and reverse primers (nucleotide sequences as shown in SEQ ID NO.5);

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

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

[0054] (2) Double digestion of some recombinant plasmids was performed using nucleases KpnI and XhoI; then, the amplification products, digestion products, and undigested recombinant plasmids were analyzed by 1.0% agarose gel electrophoresis. The electrophoresis results are as follows: Figure 3 As shown, lane 1 contains the recombinant plasmid extracted from the strain, lane 2 contains the product after double enzyme digestion, and lanes 3-4 contain the PCR product.

[0055] Depend on Figure 3 As can be seen, the recombinant plasmid yielded two bands after double enzyme digestion. One band was the same size as the amplification product, measuring 1518 bp, which was consistent with the size of the target gene fragment. The other band was the same size as the original empty vector plasmid, proving that this embodiment successfully constructed a recombinant expression plasmid for expressing recombinant human collagen and that it could be correctly amplified by PCR.

[0056] By expressing a His-tagged recombinant protein using a vector start codon, and then cleaving the N-terminal tag protein and other leader amino acid sequences by enterokinase after nickel column affinity chromatography, recombinant collagen composed of a simple collagen motif can be obtained.

[0057] Example 3:

[0058] In this embodiment, a prokaryotic expression system was used to express collagen from the recombinant expression plasmid constructed in Example 2. The specific steps are as follows:

[0059] Gene sequencing was performed on the recombinant plasmid obtained in Example 2. The correctly sequenced recombinant plasmid was extracted and transformed into the *E. coli* expression strain *Rosetta*. Recombinants were then screened, and the obtained recombinants were the fusion-expressed recombinant human collagen with a leader sequence. The transformed engineered bacteria were stored in 25% glycerol at -20°C. Empty *Rosetta* bacteria without the transformed plasmid were used as a blank control, and *Rosetta* bacteria containing the empty pET32a(+) plasmid were used as a negative control. The cultures were incubated at 37°C and 220 rpm with shaking until the OD value reached 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.05 mM, and the cultures were induced for 4 h. The bacterial cells were collected, resuspended, lysed, centrifuged, and analyzed by electrophoresis. The results are as follows: Figure 4 As shown, lane 1 is the IPTG-induced recombinant Rosetta bacteria group, lane 2 is the un-IPTG-induced recombinant Rosetta bacteria group, lane 3 is the negative control group, and lane 4 is the Rosetta empty bacteria group, i.e., the blank control group.

[0060] Depend on Figure 4 As can be seen, compared with the blank control, negative control and uninduced recombinant engineered bacteria, the induced recombinant engineered bacteria showed obvious expression of fusion protein with a molecular weight of approximately 63.4 kDa.

[0061] Example 4:

[0062] This embodiment describes the isolation and purification of the collagen fusion protein successfully expressed in Example 3. The specific steps are as follows.

[0063] The fermentation cells from Example 3 were collected, and the supernatant was obtained after lysis. Imidazole stock solution was added to make the supernatant contain a final concentration of 20 mM imidazole. The mixture was then filtered through a 0.45 μm filter membrane. Using an AKTA Pure chromatography system, a 1 mL His Trap pre-packed column was equilibrated with 20 mM PB + 0.5 M NaCl + 20 mM imidazole at pH 7.4. The sample was then loaded onto the pre-packed column. The column was reequilibrated with 20 mM PB + 0.5 M NaCl + 20 mM imidazole at pH 7.4 until the UV curve returned to baseline. The target protein was then eluted with 20 mM PB + 0.5 M NaCl + 500 mM imidazole at pH 7.4, and the elution peak was collected. For the eluted samples, dialysis desalting was performed using 50 mM Tris at pH 7.5, followed by filtration through a 0.22 μm filter membrane. Using an AKTA Pure chromatography system, a 1 mL EzFast SP FF pre-packed column was used. After equilibration with 50 mM Tris at pH 7.5, the sample was loaded. The column was reequilibrated with 50 mM Tris at pH 7.5 until the UV curve returned to baseline. The target protein was then eluted with 50 mM Tris + 1 M NaCl at pH 7.5. The elution peak was collected, and the collected samples were analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 5 As shown, lane 1 is the bacterial supernatant, lane 2 is the NI affinity chromatography flow-through peak, lane 3 is the NI affinity chromatography elution peak, lane 4 is the ion exchange SP flow-through peak, lane 5 is the ion exchange elution peak 1, lane 6 is the ion exchange elution peak 2, and lane 7 is the sample after dialysis concentration adjustment.

[0064] After dialysis and buffer replacement, the protein concentration was adjusted to 0.5 mg / ml. The protein was digested with 1 IU / mg recombinant bovine enterokinase at 20°C for 16 h. The protein was then subjected to nickel column affinity chromatography again. The flow-through was collected, concentrated by dialysis with PBS, and the results were analyzed by SDS-PAGE electrophoresis. Figure 6 As shown, lane 1 is the protease digestion product; lane 2 is the NI affinity chromatography flow-through peak, which is the purified recombinant human collagen; lane 3 is the NI affinity chromatography elution peak, which contains bands such as tag proteins.

[0065] Then, the endotoxin-removing column (Detoxi-Gel) was used for further removal of endotoxins and microorganisms.

[0066] An acidic solution of collagen monomers was dissolved in PBS buffer (pH 7.4). The instantaneous increase in pH caused the collagen to self-assemble into a high-density fibrous matrix, which was then prepared into a lyophilized collagen powder using a conventional lyophilization process.

[0067] Example 5:

[0068] The collagen lyophilized powder obtained in Example 4 was dissolved in an appropriate buffer solution, filtered through a 0.22 μm filter, and then its purity was determined using high-performance liquid chromatography (HPLC). The HPLC conditions were set as follows: mobile phase: 50 mM PB + 0.3 MnAcl, pH 6.8, flow rate: 0.3 mL / min, column temperature: 25 °C, dual-wavelength detection at 220 nm and 280 nm. The chromatogram is shown below. Figure 7 As shown, it exhibits a single peak shape and a purity higher than 95%.

[0069] Example 6:

[0070] This embodiment uses in vitro experiments to detect the in vitro cell repair ability of the recombinant human collagen from Example 1. The specific steps are as follows.

[0071] First, use a marker to mark each hole on the back of the 6-hole board by drawing three equal lines horizontally and vertically. Mark each hole approximately (5~15) × 10 mm. 5 Three T3 cells were seeded in each well, with the goal of achieving 95%–100% confluence after 24 hours of culture; each group had three replicate wells. Using a marker pen and a ruler, three horizontal lines were drawn on the bottom of each well. After 24 hours of cell culture, a 10 μL pipette tip, aligned vertically with the ruler, was used to gently push downwards along the longitudinal lines to create scratches. The cells were washed three times with PBS to remove any scratched cells. 2 mL of serum-free medium was added to the sample group and 2 mL to the control group (prepared as test sample and control material), respectively, to a final concentration of 0.5 mg / mL. The blank control group received only serum-free medium.

[0072] The cells were incubated at 37℃ in a 5% CO2 incubator. After 0h and 24h, photographs were taken under a 40x microscope, using the intersection of the horizontal and vertical streaks as the focal point. Figure 8 As shown in Table 1, the area of ​​the scratched region was measured, and the cell migration rate of each group was calculated by dividing the total area of ​​migrating cells in the fixed scratched region by the initial area of ​​the fixed scratched region. The experimental results are shown in Table 1.

[0073] Table 1. Results of scratch area and healing rate testing

[0074]

[0075]

[0076]

[0077] Table 1 shows the scratch area size and healing rate at different time points for each group. The scratch healing area is calculated as: (average scratch area at 0h) - (corresponding scratch area). The scratch healing rate is calculated as: (scratched healing area) / (average scratch area at 0h). The scratch area is represented by pixels obtained from histogram data in Photoshop. The results show that cell cultures treated with the recombinant human collagen provided by this invention and the positive control (commercially available collagen product) significantly promote cell migration. In particular, in the experimental system considered, the recombinant human collagen provided by this invention significantly enhanced cell migration compared to the positive control.

[0078] In summary, this invention constructs a novel recombinant human collagen peptide by using a self-designed sequence SEQ ID NO.1 as the motif, employing 12-23 tandem repeats as the N-terminal sequence, and using the sequence shown in SEQ ID NO.2 derived from human type III collagen as the C-terminal sequence. The recombinant human collagen peptide constructed by this invention can correctly express active collagen with a triple helix structure in a prokaryotic expression system, greatly reducing the production cost of active human collagen and improving production efficiency. Furthermore, the prepared recombinant human collagen peptide has better water retention and anti-degradation capabilities than natural collagen, and can promote cell migration and growth.

[0079] This invention has explored the optimal expression host and fermentation conditions for expressing the above-mentioned recombinant human collagen peptides, and obtained high expression in the fermentation broth, which is far superior to the eukaryotic expression system and can be used for large-scale production.

[0080] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A recombinant human collagen-like polypeptide, characterized in that: The recombinant human collagen polypeptide consists of an N-terminal repeat sequence tandemly 16 times and a C-terminal sequence; the repeat sequence is shown in SEQ ID NO.1; the C-terminal sequence is shown in SEQ ID NO.

2.

2. A gene encoding the recombinant human collagen polypeptide of claim 1, characterized in that: The gene sequence is shown in SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that: The recombinant expression vector carries the gene sequence as described in claim 2.

4. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the recombinant expression vector as described in claim 3.

5. A method for preparing recombinant human-like collagen polypeptide, characterized in that, Includes the following steps: The recombinant engineered bacteria as described in claim 4 are fermented and cultured, the bacterial cells are collected, the bacteria are broken down, and recombinant human-like collagen polypeptides are isolated.

6. The method for preparing recombinant human collagen polypeptide as described in claim 5, characterized in that, The preparation method specifically includes the following steps: the recombinant engineered bacteria are cultured with shaking until the OD value is 0.6~0.8, induced with an inducer, the bacterial cells are collected, the bacteria are broken, and the recombinant human-like collagen polypeptide is isolated.

7. The method for preparing recombinant human collagen polypeptide as described in claim 6, characterized in that: The inducer includes isopropyl-β-D-thiogalactoside.

8. The method for preparing recombinant human collagen polypeptide as described in claim 6, characterized in that: It also includes obtaining the supernatant after lysis, adding imidazole, filtering, and using a protein purification instrument to separate and purify the recombinant human collagen peptides.

9. The use of the recombinant human collagen polypeptide as described in claim 1 in the preparation of collagen gels or collagen injections.

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