Recombinant human-like collagen polypeptide with a stable fragment derived from human ColⅢ, preparation method and uses thereof

Abstract

The present disclosure belongs to the field of biomedicine and relates to a recombinant human-like collagen polypeptide with a stable fragment derived from human ColⅢ, a preparation method thereof, and uses thereof. The present disclosure constructs a new recombinant human-like collagen polypeptide by intercepting the ColⅠα1 Gly485-Pro514 sequence (GER GGPGSRGFPGADGVAGPKGPAGERGSP) of human type I collagen as a motif, taking 10-25 tandem repeats as the N-terminal sequence, and using the sequence GPPGAPGPCCGGV derived from human type III collagen as the C-terminal sequence. The recombinant human-like collagen polypeptide constructed in the present disclosure can correctly express active collagen with a triple helix structure in a prokaryotic expression system, reducing the production cost of active human-derived collagen and improving the production efficiency, and the prepared recombinant human-like collagen polypeptide has good skin repair ability.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and relates to a recombinant human-like collagen polypeptide with a stable fragment derived from human ColⅢ, a preparation method thereof, and uses thereof. Background Art

[0002] Type I collagen (Collagentype I, Col I) is the only component that makes up the collagen fibers in the dermis, accounting for more than 80% of the skin; it is the most abundant component in dermal connective tissue. Under the epidermis, the collagen fibers near epidermal appendages and blood vessels are thin and have no definite orientation. The collagen fibers in other parts of the dermis are all combined into bundles. The collagen bundles are thinner in the upper part of the dermis and thicker in the lower part. In the middle and lower parts of the dermis, the direction of the collagen bundles is almost parallel to the skin surface and intersect with each other, extending in various directions on a horizontal plane. Collagen fibers are considered to be the dermal formed elements most closely related to skin aging.

[0003] Type I collagen is a heterotrimeric helix molecule. Each chain consists of more than 1000 amino acids. The length of the type I collagen molecule is about 300 nm, and the width is about 1 - 5 nm. The collagen triple helix has a very high tensile strength. In most cases, it consists of two α1 chains and one α2 chain, while the α1 homotrimer helix exists in a minor form.

[0004] Type I collagen provides the functions of supporting and maintaining elasticity for the skin. As people age, the content of type I collagen gradually decreases, making the skin become loose and develop more wrinkles. Therefore, by injecting type I collagen or using skin care products containing type I collagen, the elasticity of the skin can be increased, and the appearance of wrinkles can be reduced. Type I collagen can be used to fill fine facial wrinkles, drooping corners of the mouth, lip defects, periorbital depression, dark circles under the eyes, etc. Compared with traditional filling materials, type I collagen can provide longer-lasting effects and is more natural. It can also be used for hydrodermabrasion injection. It can improve the skin quality and make the skin more translucent. The principle is to inject type I collagen on the skin surface to make the skin more moist and smooth. At the same time, it can promote the regeneration and repair of skin cells, and in the case of damaged or aged skin, reduce skin relaxation and wrinkles.

[0005] Currently, type I collagen can be obtained from the skin, bones, scales and other parts of aquatic products such as fish. There are certain differences between mammalian-derived collagen and aquatic-derived collagen, which are manifested in physical and chemical properties, chemical composition, solution properties, etc. From the perspective of homology, using human-derived type I collagen is the best in terms of beauty effects and anti-rejection.

[0006] However, the monomer molecular weight of human type I collagen is relatively large, and it has a complex active structure. It is difficult to express the full-length protein from a prokaryotic expression system, while the eukaryotic expression system has the disadvantages of low expression level and high separation and purification cost. Using truncated human type I collagen polypeptide is a current research hotspot. However, it is a very difficult task to determine which sequence segment to truncate and how long the truncated sequence should be so that it can both preserve the activity of the wild protein and be efficiently expressed in the prokaryotic expression system. Summary of the Invention

[0007] In view of the above problems, the present disclosure discloses a recombinant human-like collagen polypeptide, a preparation method thereof, and uses thereof.

[0008] The present disclosure uses the sequence of Gly485 - Pro514 of Col Iα1 (SEQ ID NO: 1 GERGGPGSRGFPGADGVAGPKGPAGERGSP) as a motif, performs several tandem repeats, and uses the SEQ ID NO: 2 GPPGAPGPCCGGV from human type III collagen (Collagen type I, Col III) as the C-terminal sequence to reconstruct a recombinant human-like collagen polypeptide. This polypeptide not only retains the biological activity of collagen but also can be correctly and highly expressed in the prokaryotic expression system.

[0009] The present disclosure includes the following technical solutions:

[0010] In a first aspect, the present disclosure provides a recombinant human-like collagen polypeptide, which includes a tandem repeat sequence at the N-terminal and a C-terminal sequence; the repeat sequence is as shown in SEQ ID NO: 1; the C-terminal sequence is as shown in SEQ ID NO: 2.

[0011] Further, the number of repeats of the repeat sequence is between 10 and 25 times, preferably 16 times.

[0012] In a second aspect, the present disclosure also discloses a biological material, including any one of (i) - (iii):

[0013] (i) A nucleic acid molecule encoding the recombinant human-like collagen polypeptide according to the first aspect of the present disclosure;

[0014] (ii) A recombinant expression vector, a recombinant plasmid containing the nucleic acid molecule in (i);

[0015] (iii) A transformant, a host cell containing the nucleic acid molecule in (i) or the recombinant expression vector in (ii).

[0016] Further, the original plasmid of the recombinant plasmid is pET32a(+), and the host cell is Rosetta strain.

[0017] In a third aspect, the present disclosure also discloses a method for preparing the recombinant human collagen polypeptide described in the first aspect, including expressing the (i) nucleic acid molecule or (ii) recombinant expression vector described in the second aspect of the present disclosure to obtain the recombinant human collagen polypeptide; alternatively, culturing the (iii) transformant described in the second aspect of the present disclosure and isolating the recombinant human collagen polypeptide; IPTG (isopropyl-β-D-thiogalactoside) is preferably added during the expression or culturing process.

[0018] In a fourth aspect, the present disclosure also discloses the application of the recombinant human collagen polypeptide described in the first aspect or the recombinant human collagen polypeptide prepared by the preparation method described in the third aspect in the preparation of a collagen gel or a collagen injection.

[0019] Furthermore, the above-mentioned collagen injection is used for preparing beauty products.

[0020] In a fifth aspect, the present disclosure discloses a collagen injection containing the recombinant human collagen polypeptide described in the first aspect.

[0021] Furthermore, the above-mentioned collagen injection further contains an auxiliary active functional molecule; the auxiliary active functional molecule includes ferrous gluconate and / or botulinum toxin type A.

[0022] Technical effects achieved by the present disclosure:

[0023] By intercepting the ColⅠα1 Gly485-Pro514 sequence (GERGGPGSRGFPGADGVAGPKGPAGERGSP) of human type I collagen as a motif, taking 10 to 25 tandem repeats (preferably 16 times) as the N-terminal sequence, and using GPPGAPGPCCGGV derived from human type III collagen as the C-terminal sequence, the present disclosure constructs a new recombinant human collagen polypeptide. The recombinant human collagen polypeptide constructed by the present disclosure can correctly express active collagen with a triple helix structure in a prokaryotic expression system, greatly reducing the production cost of active human-derived collagen and improving production efficiency, and the prepared recombinant human collagen polypeptide has excellent skin repair ability.

[0024] The present disclosure has explored the fermentation conditions for expressing the above-mentioned recombinant human collagen polypeptide in a prokaryotic expression host, obtained a high expression of the recombinant human collagen polypeptide, which is far superior to the eukaryotic expression system and can be used for large-scale production.

[0025] The recombinant human-like collagen polypeptide described in the present disclosure is prepared into a collagen gel or an injectable collagen polypeptide, which can be used in the field of medical aesthetics to prepare beauty products or skin repair products for repairing skin trauma, eliminating skin wrinkles, and keeping the skin smooth and elastic for a long time. When made into an injection, compared with the prior art that requires injection once every 6 - 12 months under the same conditions, the injection frequency can be extended to once every 18 - 24 months, reducing the injection frequency and improving the compliance of patients / customers. Description of the Drawings

[0026] Figure 1 It is the prediction result of the triple helix structure of recombinant human-like collagen 1 in Example 1;

[0027] Figure 2 It is the prediction result of the triple helix structure of recombinant human-like collagen 2 in Example 1;

[0028] Figure 3 It is the plasmid map of plasmid pET32a+ used in Example 2;

[0029] Figure 4 It is the identification result of recombinant plasmid PCR and enzyme digestion;

[0030] Figure 5 It is the separation and purification result of the protein induced and expressed by recombinant bacteria;

[0031] Figure 6 It is the identification result of recombinant protein and target protein;

[0032] Figure 7 It is the result of the scratch experiment of the blank control group;

[0033] Figure 8 It is the result of the scratch experiment of recombinant protein 1;

[0034] Figure 9 It is the result of the scratch experiment of marine collagen. Detailed Embodiments

[0035] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and the laboratory operation steps used herein are all terms widely used in the corresponding fields and conventional steps. At the same time, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0036] The term "and / or" means any one of the options or two or more of the options.

[0037] The term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this text, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the stated elements, integers or steps is also covered.

[0038] Example 1

[0039] In this example, the amino acid sequence of recombinant human-like collagen was designed and its spatial conformation was predicted.

[0040] 1.1 The amino acid sequence of recombinant human-like collagen involves

[0041] In this example, the Col Iα1 Gly485 - Pro514 sequence of human type I collagen (GERGGPGSRGFPGADGVAGPKGPAGERGSP, SEQ ID NO: 1) was intercepted as a motif, and 16 tandem repeats were taken, and then the sequence GPPGAPGPCCGGV (SEQ ID NO: 2) derived from human type III collagen was connected to the C-terminus to obtain recombinant human-like collagen 1.

[0042] 1.2 Prediction of the spatial conformation of recombinant human-like collagen

[0043] Using the online tool: https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / to predict the structure of the recombinant human-like collagen 1 designed above, the result is as Figure 1 shown, the recombinant human-like collagen 1 designed in this example can form a good triple helix structure.

[0044] 1.3 On the basis of step 1.1, the C-terminal sequence was replaced with the sequence GPPGPAGANGAPG (SEQ ID NO: 3) which is also derived from human type III collagen but different from SEQ ID NO: 2 to obtain a new recombinant human-like collagen 2, and then step 1.2 was repeated to predict the structure of this protein 2, and the result is as Figure 2 shown, this protein 2 cannot form a triple helix structure, from which it can be inferred that the selected C-terminal sequence in this example has an important influence on the stability of the triple helix structure of recombinant human-like collagen.

[0045] Example 2

[0046] In this example, for the purpose of expressing the recombinant human-like collagen 1 designed in Example 1, Nanjing Genscript Biotech Co., Ltd. was commissioned to construct a recombinant expression plasmid for expressing recombinant human-like collagen using plasmid pET32a(+) as the original plasmid (the plasmid map is as Figure 3 shown).

[0047] 2.1 Design of Inserted Nucleic Acid Molecule

[0048] The amino acid sequence of recombinant human-like collagen 1 designed in Example 1 was decoded to obtain the corresponding nucleotide sequence, and the codons were optimized for E. coli expression. Then, a nucleotide sequence encoding the restriction enzyme cleavage site of KpnΙ (GGTACC) and a leader sequence (GACGATGATGATAAA, SEQ ID NO: 4) were added successively at the 5' end, and a nucleotide sequence encoding two stop codons (TAATGA) and a nucleotide sequence encoding the restriction enzyme cleavage site of Xho I (CTCGAG) were added at the 3' end to facilitate cloning into the pET32a(+) expression vector. The resulting nucleic acid molecule had a full length of 1512 bp (as shown in SEQ ID NO: 5).

[0049] 2.2 Construction of Recombinant Expression Plasmid

[0050] The nucleic acid molecule encoding recombinant human-like collagen designed in step 2.1 was synthesized by chemical synthesis. The synthesized nucleic acid molecule and the plasmid pET32a(+) were digested with the restriction enzymes KpnI and XhoI respectively, and then a ligation product containing the recombinant plasmid was obtained under the action of a ligase. The ligation product was transformed into the DH5α E. coli cloning strain and cultured at 37°C on an LB resistant medium containing 100 μg / mL ampicillin (Amp) to screen for recombinants. The recombinant plasmid obtained by culturing was extracted and subjected to the following treatments: (1) PCR amplification of part of the recombinant plasmid using a forward primer (nucleotide sequence as shown in SEQ ID NO: 4) and a reverse primer (nucleotide sequence as shown in SEQ ID NO: 5); (2) double digestion of part of the recombinant plasmid with the restriction enzymes KpnI and XhoI; and then the amplified product, the digested product, and the undigested recombinant plasmid were analyzed by 0.8% agarose gel electrophoresis. The electrophoresis results are as Figure 4 shown, where lane 1 is the PCR amplification product, lane 2 is the undigested recombinant plasmid, and lane 3 is the digested product. As Figure 4 can be seen, two bands were obtained after double digestion of the recombinant plasmid. One band was the same size as the amplified product, with a size of 1512 bp, which was the target gene fragment; the other band was the same size as the original plasmid, proving that the recombinant expression plasmid for expressing recombinant human-like collagen was successfully constructed in this example.

[0051] SEQ ID NO: 5:

[0052]

[0053] Example 3

[0054] In this example, the prokaryotic expression system was used to express collagen from the recombinant expression plasmid constructed in Example 2. The specific steps are as follows:

[0055] The recombinant plasmid obtained in Example 2 was subjected to gene sequencing. The recombinant plasmid with correct sequencing identification was extracted and transformed into the Escherichia coli expression strain Rosetta. Then, recombinants were screened, and the obtained recombinants were recombinant human-like collagen with a leader sequence expressed by fusion. The transformed engineering bacteria were stored in 25% glycerol and frozen at -20°C in a 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. After bacterial culture and IPTG-induced expression, the culture conditions were as follows: cultured with shaking at 37°C until the OD value reached 0.7, IPTG was added to a final concentration of 0.01 mM, and induced for 4 h. The bacterial cells were collected, resuspended, lysed, centrifuged, and detected by electrophoresis. The results are as Figure 5 shown. Lane 1 is the Rosetta empty bacteria, that is, the blank control group, lane 2 is the negative control group, lane 3 is the recombinant Rosetta bacteria group without IPTG induction, and lane 4 is the recombinant Rosetta bacteria group induced by IPTG. As Figure 5 can be seen, compared with the blank control, negative control, and uninduced recombinant bacteria, the induced recombinant bacteria had obvious fusion protein expression, and the protein molecular weight was about 61.5 kDa.

[0056] Example 4

[0057] In this example, the collagen fusion protein successfully expressed in Example 3 was separated and purified and prepared into a powder. The specific steps are as follows:

[0058] The fermented bacterial cells in Example 3 were collected. After lysis, the supernatant was obtained. Imidazole mother liquor was added to make the supernatant contain imidazole with a final concentration of 40 mM, and filtered through a 0.45 m filter membrane. Using an AKTA Pure chromatography system and a 1 mL HisTrap pre-packed column, after equilibrating the pre-packed column with 20 mM PB + 0.5 M NaCl + 40 mM imidazole, pH 7.4, the sample was loaded. After re-equilibrating with 20 mM PB + 0.5 M NaCl + 40 mM imidazole, pH 7.4 until the UV curve dropped 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. After dialysis to change the buffer, the protein concentration was adjusted to 0.5 mg / ml, and it was digested with 1 IU / mg recombinant bovine enterokinase at 20°C for 16 h. After nickel column affinity chromatography again, the flow-through was collected, concentrated by PBS dialysis, and the SDS-PAGE electrophoresis detection results are as Figure 6As shown, lane 1 is the fusion protein (elution peak of nickel column affinity chromatography), and lane 2 is the target protein (the final product after removing the leading sequence by enzymatic digestion).

[0059] Collect the target protein collagen monomer, and then use the Detoxi-Gel endotoxin removal column to further remove endotoxins and microorganisms. Mix the acidic solution of the collagen monomer with PBS buffer (pH 7.4). The self-assembly of collagen into a high-density fiber matrix is induced by a transient increase in pH value, and then the collagen freeze-dried powder is prepared according to the conventional freeze-drying process.

[0060] Example 5

[0061] In this example, an in vitro experiment was used to detect the in vitro cell repair ability of the recombinant human-derived collagen 1 obtained in Example 1.

[0062] In this example, in a 12-well plate, human skin keratinocytes were cultured in DMEM medium (Dulbecco's Modified Eagle’s medium) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2 to obtain a single cell layer of human skin keratinocytes. Then, an artificial scratch was created by sliding the pipette tip on the single cell layer. Then, DMEM cell culture medium containing 0.1% (g / mL) recombinant human-derived collagen 1 was added to the artificial scratch, and the repair of the artificial scratch was detected at 2 h (T2), 8 h (T8), and 24 h (T24) later. In the experiment, the untreated DMEM medium was used as the blank control group (CTR), and marine collagen (0.1% g / mL of marine collagen) was used as the positive control group.

[0063] At each experimental time, the single cell layer was visually inspected at 4× magnification using a camera (OPTIKA, XDS-2), and camera pictures were collected (TiEsseLab PrimoCamHD 5.0 with sensor micron MT9P001, and the pictures were analyzed using Image J software ( Figures 7 to 9 as shown). The distance between the wound edges of each picture was measured in μm and recorded at 3 points on the wound length. The t-test was used to analyze the changes in the wound edges.

[0064] The experimental results are as follows:

[0065] Table 1 Detection results of scratch width

[0066]

[0067] Table 2 Percentage of scratch width of each experimental group relative to the initial width

[0068]

[0069] The data in Table 1 represent the distance between the wound edges (μm) (expressed as mean ± standard deviation). The data in Table 2 represent the percentage change in the distance between the wound edges compared to the initial scratch width for treated and untreated cells at each experimental time. According to the results obtained, it can be emphasized that the cell cultures treated with 0.1% of recombinant human collagen 1 and marine collagen significantly promoted wound repair by being faster. In particular, in the experimental system considered, recombinant human collagen 1 had a better repair performance than marine collagen at all experimental times (p value < 0.05 at T0, T2, T8, T24).

[0070] It can be seen from the above embodiments and test examples that the present invention constructs a new recombinant type I human collagen polypeptide by intercepting the COL1a1Gly485-Pro514 sequence (GERGGPGSRGFPGADGVAGPKGPAGERGSP) of human type I collagen as a motif, taking 10-25 times (preferably 16 times) of tandem repeats as the N-terminal sequence, and using GPPGAPGPCCGGV derived from human type III collagen as the C-terminal sequence. The recombinant type I human collagen polypeptide constructed in the present invention can correctly express the 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, and the prepared recombinant human collagen polypeptide has significant skin repair ability.

[0071] The present disclosure explores the expression host and fermentation conditions for expressing the above-mentioned recombinant type I human collagen polypeptide, and obtains high expression in the fermentation broth, which is far superior to the eukaryotic expression system and can be used for large-scale production.

[0072] The recombinant human type I collagen polypeptide disclosed in the present invention is used to prepare collagen gel or recombinant human type I collagen polypeptide for injection, which can be used in the field of medical cosmetology to repair skin trauma, eliminate skin wrinkles, and keep the skin smooth and elastic for a long time. When it is made into an injection, under the same conditions, compared with the prior art of once every 6-12 months, the injection period can be extended to once every 18-24 months, thereby reducing the injection frequency and improving patient / customer compliance.

[0073] The above are only a few preferred embodiments of the present disclosure, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present disclosure. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present disclosure, which all belong to the protection scope of the present disclosure.

Claims

1. A recombinant human collagen polypeptide, characterized in that: The amino acid sequence of the recombinant human collagen polypeptide consists of a 16-times tandem repeat sequence of SEQ ID NO: 1 at the N-terminus and a C-terminal sequence of SEQ ID NO:

2.

2. Biological material, including any one of (i) to (iii): (i) a nucleic acid molecule encoding the recombinant human collagen polypeptide according to claim 1; (ii) a recombinant expression vector, comprising a recombinant plasmid of the nucleic acid molecule of (i); (iii) Transformants, host cells containing the nucleic acid molecule (i) or the recombinant expression vector (ii).

3. The biomaterial according to claim 2, characterized in that The original plasmid of the recombinant plasmid is pET32a (+), and the host cell is a Rosetta strain.

4. The method for preparing the recombinant human-like collagen polypeptide according to claim 1, characterized in that: The preparation method comprises expressing the nucleic acid molecule (i) as described in claim 2 to obtain a recombinant human-like collagen polypeptide; or, expressing the recombinant expression vector (ii) as described in claim 2 to obtain a recombinant human-like collagen polypeptide; or, culturing the transformant (iii) as described in claim 2 and isolating and obtaining the recombinant human-like collagen polypeptide; and adding IPTG during the expression or culturing process.

5. Use of the recombinant human-like collagen polypeptide according to claim 1, or the recombinant human-like collagen polypeptide obtained by the preparation method according to claim 4 in the preparation of collagen gel or collagen injection.

6. The use according to claim 5, characterized in that: The collagen injection is used for preparing beauty products or skin repair products.

7. A collagen injection, characterized in that: The collagen injection contains the recombinant human-like collagen polypeptide according to claim 1.

8. The collagen injection according to claim 7, characterized in that: It also contains auxiliary active functional molecules; the auxiliary active functional molecules include ferrous gluconate and / or botulinum toxin type A.

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