Recombinant humanized type iii collagen having triple helix structure and use thereof

Abstract

The present invention relates to the technical field of protein engineering and provides a recombinant humanized type III collagen having a triple helix structure, a preparation method therefor, and a use thereof. The amino acid sequence of the recombinant humanized type III collagen comprises n core units, wherein n is 1 or an integer greater than 1, and the amino acid sequence of the core unit is as shown in SEQ ID NO: 1. The present invention further provides a method for constructing an expression system and a method for preparing a recombinant humanized type III collagen. The present invention further provides an actual industrial use on the basis of the characteristics of the protein. The recombinant humanized type III collagen provided by the present invention has high purity, good stability, and high biological activity. Experiments have shown that the recombinant humanized type III collagen has various biological activities such as promoting cell proliferation and migration.

Description

Recombinant humanized type III collagen with triple helix structure and its application Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to recombinant humanized type III collagen with a triple helix structure and its applications. Background Technology

[0002] Collagen accounts for 25%–30% of the total protein in the human body. Of the 28 types of collagen, types I, II, and III account for 90%. Human type III collagen is a fibrous protein formed by three alpha helical chains. Mature type III collagen contains 1068 amino acids per protein chain, and the three chains form a triple helix with a molecular weight close to 400 kDa. When used as an ingredient in skincare products, collagen's natural micellar structure and large molecular weight make it difficult to penetrate the skin barrier and reach the dermis to exert its effects. Type III collagen contains multiple signaling pathway recognition sites, such as integrin recognition sites, which activate collagen regeneration. The amino acid sequence of type III collagen is a repetitive sequence Gly-XY, where X and Y can be any amino acid, but X is usually proline (Pro), and Y is usually hydroxyproline (Hyp) and hydroxylysine (Hyl). Hyp and Hyl require modification with hydroxylases.

[0003] It has been reported that triple-helix collagen exhibits better stability, biocompatibility, and cell proliferation activity than single-chain collagen. However, the formation of the triple-helix structure in recombinant collagen currently relies mainly on the matching between the sequence and hydroxylases from different sources, and there are few reports of recombinant collagen that can autonomously assemble into a triple helix. Summary of the Invention

[0004] The primary objective of this invention is to provide a recombinant humanized type III collagen with a smaller molecular weight than natural collagen and a triple-helix structure. This invention obtains the core sequence of human type III collagen by screening the mature region sequence and, through repetition, obtains a sequence capable of self-assembling into a triple helix in vitro.

[0005] The amino acid sequence of the recombinant humanized type III collagen core sequence of the present invention has more than 90% homology with the sequence shown in SEQ ID No. 1, that is, the sequence homology can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%; preferably, the amino acid sequence of the recombinant humanized type III collagen core sequence is as shown in SEQ ID No. 1.

[0006] Regarding amino acid sequence homology, on the one hand, it is based on conserved amino acid substitution. This conservation ensures that structurally related amino acids can similarly substitute for certain amino acids without significantly affecting the properties of the resulting variant polypeptides. For example, an amino acid residue may be substituted by an amino acid residue with a similar side chain. On the other hand, it considers adding one or more redundant sequences to SEQ ID No. 1 without resulting in a significant increase in biological activity or structural stability. That is, the original biologically active fragment does not perform its biological function, such as its ability to bind to receptors, better due to the addition of redundant sequences. Therefore, amino acid changes falling within the scope of this invention are achieved by selecting changes that significantly enhance the maintenance of biological functions.

[0007] The recombinant humanized type III collagen core sequence described in this invention is repeatedly tandemly repeated to form a triple helix sequence that can self-assemble in vitro.

[0008] Preferably, the number of repetitions is 4-16; more preferably, the number of repetitions is 7, 8, or 9; and most preferably, the number of repetitions is 8.

[0009] The repeated tandem connection can be a series connection of core units head to tail, or it can be a series connection of core units by a linker. The linker preferably contains glycine and / or serine, and preferably contains 1-5 amino acids.

[0010] The recombinant collagen of this invention may carry a tag consisting of one or more histidine residues. The tag is attached to the N-terminus or C-terminus of the recombinant collagen. The tag can be used to purify the protein using a chromatographic method with a nickel-based chromatographic medium.

[0011] More preferably, the recombinant humanized type III collagen is obtained by retaining two amino acids, Ala and Lys (corresponding to positions 286-287 of the maturation region of human type III collagen), at the N-terminus, which can further improve the structural stability of the target protein.

[0012] Most preferably, the amino acid sequence of the recombinant humanized type III collagen of the present invention is shown in SEQ ID No. 2. This sequence is formed by repeating the core sequence (SEQ ID No. 1) eight times in tandem, and retaining the two amino acids Ala and Lys at the N-terminus.

[0013] For the mature region of human type III collagen, please refer to NCBI RefSeq:NP_000081.2, mat_peptide 154..1221, / product="collagenalpha-1(III)chain".

[0014] The present invention provides a gene encoding the recombinant humanized type III collagen, wherein the nucleotide sequence of the gene has more than 80% homology with the nucleotide sequence of SEQ ID No. 3; preferably, the nucleotide sequence of the gene has more than 90% homology with the nucleotide sequence of SEQ ID No. 3; most preferably, the nucleotide sequence of the gene is as shown in SEQ ID No. 3.

[0015] Regarding nucleotide sequence homology, on the one hand, due to the degeneracy of codons, the nucleotide sequence encoding a protein is not unique. Therefore, any nucleotide sequence that can encode the amino acid sequence of the recombinant humanized type III collagen of this invention is within the scope of protection of this invention.

[0016] The present invention provides a recombinant vector for expressing the recombinant humanized type III collagen, comprising an initial vector and the gene.

[0017] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising one or more nucleic acid molecules that are operatively linked. Vectors may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome.

[0018] The present invention provides recombinant cells expressing the recombinant humanized type III collagen, wherein the recombinant cells are transfected with the recombinant vector.

[0019] The recombinant cells are preferably Pichia pastoris GS115.

[0020] A second objective of this invention is to provide a method for expressing the recombinant humanized type III collagen, preferably a method for expression in Pichia pastoris GS115, comprising the following steps:

[0021] i) The target fragment is cloned and assembled into a vector plasmid, transformed into E. coli JM109 for amplification, and the plasmid is extracted;

[0022] ii) Linearize plasmid pPICZαA with the rapid digestion enzyme SacI, electrotransform it into Pichia pastoris GS115, and perform primary and secondary screening using YPD resistance plates;

[0023] iii) Pick the transformants into BMGY medium, culture them, centrifuge and discard the supernatant; resuspend the bacterial cells in BMMY medium and induce culture; the culture medium is then separated and purified to obtain the target protein.

[0024] Preferably, using YPD resistance plates for initial screening and secondary screening means using 100 μg / mL YPD resistance plates for initial screening and 800 μg / mL YPD resistance plates for secondary screening.

[0025] Preferably, the induction culture is carried out using 1% methanol, which is added every 24 hours for a total of 96 hours.

[0026] A third object of the present invention is to provide the recombinant type III collagen, wherein the recombinant type III collagen prepared by the preparation method comprises any of the following applications:

[0027] i) Promotes cell proliferation;

[0028] ii) Promotes cell migration;

[0029] iii) Preparation of cosmetics;

[0030] iv) Preparation of medical materials.

[0031] The cosmetics mentioned include, but are not limited to, skin lotion, skin cream, essence water, face mask, scalp essence, and shampoo.

[0032] The medical materials mentioned include, but are not limited to, filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug delivery carriers, wherein the repair materials include, but are not limited to, bone repair materials, wound dressings, and sutures.

[0033] This invention also provides a composition comprising at least one cosmetically or pharmaceutically effective amount of the aforementioned protein and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant. In some embodiments, the dosage form of the composition includes, but is not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or freeze-dried products; furthermore, to promote transdermal absorption of collagen molecules, a patch product can be prepared using a solid carrier such as nonwoven fabric and applied to the face to prolong the interaction time between the collagen liquid and the skin surface. The recombinant type III collagen of this invention can be prepared and used in tandem with recombinant or natural type I, type II, type III, type IV, type V, type XVII collagen, fibronectin, human epidermal growth factor, and other bioactive ingredients, or can be mixed with the above-mentioned bioactive ingredients after separate preparation. The composition also contains additives such as stabilizers, preservatives, and carriers. The additives include, but are not limited to, liposomes, biodegradable microcapsules, aerosols, powders, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexanediol, and tocopherol.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The recombinant type III collagen of this invention can self-assemble into a stable triple helix structure in vitro and has good biological activity in promoting cell proliferation and migration. Attached Figure Description

[0036] Figure 1 is an SDS-PAGE image of the recombinant type III collagen LY01 of the present invention.

[0037] Figure 2 is an SDS-PAGE image of the recombinant type III collagen LY02 of the present invention.

[0038] Figure 3 is an SDS-PAGE image of the recombinant type III collagen LY03 of the present invention.

[0039] Figure 4 shows the circular dichroism (CD) spectrum of the recombinant type III collagen LY01 of this invention.

[0040] Figure 5 shows the circular dichroism (CD) chromatogram of the recombinant type III collagen LY02 of this invention.

[0041] Figure 6 shows the results of the cell proliferation experiment of the recombinant type III collagen LY01 of the present invention.

[0042] Figure 7 shows the cell scratch test results of the recombinant type III collagen LY01 of the present invention.

[0043] Figure 8 shows the confluence of cells in the scratch assay of the recombinant type III collagen LY01 of this invention. Detailed Implementation

[0044] In this invention, the gene can be synthesized by a biotechnology company. This invention does not specifically limit the method of separation and purification; conventional protein separation and purification methods in the art are acceptable. Preferred technical solutions are described in the embodiments.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1: SDS-PAGE experiment of recombinant type III collagen LY01 of the present invention

[0047] The recombinant type III collagen LY01 of this invention has the following specific amino acid sequence:

[0048] First, the nucleic acid fragment encoding LY01 (SEQ ID No. 3) was synthesized by Genscript Biotech Inc. The target fragment was then cloned and assembled into the vector plasmid pPICZαA in one step, transformed into E. coli JM109, amplified, and the plasmid was extracted.

[0049] The plasmid was linearized using the rapid digestion enzyme SacI, electroporated to GS115, and initially screened using 100 μg / mL YPD resistance plates, followed by secondary screening using 800 μg / mL YPD resistance plates.

[0050] Positive transformants were picked and cultured in BMGY medium at 30°C until OD500. 600 If the concentration is greater than 15, centrifuge and discard the supernatant; resuspend the bacterial cells in BMMY medium, and then induce culture: use 1% methanol, add it every 24 hours, and end the culture after a total of 96 hours of induction.

[0051] Separation and purification:

[0052] The fermentation broth was centrifuged at 4000g to complete solid-liquid separation. The fermentation supernatant was filtered through a 0.22μm membrane and then dialyzed overnight using a 5kDa pore size dialysis bag with 20mM, pH 5.0 PB buffer.

[0053] Next, elution was performed using a HiTrap SP HP 5ml (Cytiva) chromatography column with the following parameters:

[0054] Sample environment: 20mM PB, pH 5.0;

[0055] Buffer A (equilibration solution): 20 mM PB pH 5.0

[0056] Buffer B (elution buffer): 20 mM PB pH 5.0, 1 M NaCl;

[0057] Experimental flow rate: 4 ml / min;

[0058] Gradient elution: Elute with 0-100% buffer B, collect the elution peak at 220 nm, 15 CV.

[0059] SDS-PAGE analysis was performed on the purity and expression level of the purified protein, and the results are shown in Figure 1.

[0060] Example 2: SDS-PAGE experiment of recombinant type III collagen LY02 of the present invention

[0061] The recombinant type III collagen LY02 of this invention has the following specific amino acid sequence:

[0062] (SEQ ID No. 4). The nucleic acid fragment encoding LY02 was synthesized by GenScript. Other steps were the same as in Example 1. The results of SDS-PAGE analysis of the purified protein are shown in Figure 2.

[0063] Example 3: SDS-PAGE experiment of recombinant type III collagen LY03 of the present invention

[0064] The recombinant type III collagen LY03 of this invention has the following specific amino acid sequence:

[0065] AKGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGER (SEQ ID No. 5). The nucleic acid fragment encoding LY03 was synthesized by GenScript. Other steps were as described in Example 1. The purified protein was analyzed by SDS-PAGE, and the results are shown in Figure 3.

[0066] Example 4: Structural characterization of the recombinant type III collagen of the present invention

[0067] This invention characterizes the structure of collagen using circular dichroism (CD), a commonly used spectroscopic method in the field. CD is used to determine the structure of compounds with chiral structures that produce differential absorption between left and right rotation, and is mainly used to determine the asymmetry of molecular structures. Most biological macromolecules contain chiral groups and structures; therefore, CD is often used to measure and observe changes in the structure and conformation of biological macromolecules. The CD characteristic of the triple helix structure of collagen generally shows a positive absorption peak near 221 nm and a negative absorption peak near 195 nm. The positions of the absorption peaks shift with changes in the amino acid sequence and length.

[0068] The recombinant collagen solutions prepared in Examples 1 and 2 were subjected to CD detection. The results are shown in Figures 4 and 5. LY01 and LY02 both have a maximum characteristic positive peak at approximately 221 nm and a negative peak at less than 200 nm, verifying that the recombinant collagen of the present invention has a triple helix structure.

[0069] Example 5: Cell proliferation experiment using recombinant type III collagen LY01 of the present invention.

[0070] (1) After counting mouse embryonic fibroblasts BALB / 3T3 (Haixing Biotechnology, Cat:TCM-C714), they were seeded into 96-well cell culture plates at 2000 cells / well and cultured statically at 37℃ in a 5% CO2 cell culture incubator for 24h.

[0071] (2) The recombinant collagen solution prepared in Example 1 was diluted to a concentration of 1 μg / ml using basal culture medium. 100 μl was added to a cell culture plate. The control group was PBS, and the volume added was 2.5%. The plate was then placed in a 37°C, 5% CO2 incubator for 24 h.

[0072] (3) Prepare CCK8 working solution. Dilute CCK8 reagent with DMEM medium at a ratio of 1:10, remove the supernatant, add the prepared CCK8 working solution, and incubate at 37℃ for 2 hours. Read the absorbance of the 96-well plate at 450nm using a microplate reader and record the results (as shown in Figure 6).

[0073] In the cell proliferation experiment, after the cells in the experimental group were cultured with the recombinant collagen solution prepared in Example 1 for 24 h, the cell proliferation rate was significantly faster than that of the blank control group (PBS), indicating that recombinant type III collagen LY01 has proliferative activity on BALB / 3T3 cells.

[0074] Example 6: Cell scratch assay of recombinant type III collagen LY01 of the present invention

[0075] The cell scratch assay is a commonly used method to detect the cell migration activity of collagen; a higher migration rate reflects better biological activity of the collagen. The specific steps are as follows:

[0076] (1) After counting BALB / 3T3 cells, follow the regimen of 2×10 5 One cell per well was seeded into a 6-well cell culture plate and incubated at 37°C in a 5% CO2 incubator. Once the cell density reached 90%, lines were drawn on the 6-well plate using a yellow pipette tip, and the initial scratch position was recorded by photographing under a microscope.

[0077] (2) The recombinant collagen solution prepared in Example 1 was diluted to a concentration of 1 μg / ml using basal culture medium. 2 ml of the solution was added to a cell culture plate. The control group was natural collagen and the blank control group was PBS. The cells were then placed in a 37°C, 5% CO2 incubator and cultured for 24 h.

[0078] (3) Take photos under a microscope to record cells at the same scratch location.

[0079] (4) The scratch area at cell initiation and after 24 hours of treatment was calculated using ImageJ software, and the wound healing rate was calculated. Wound healing rate = (0h scratch area - 24h scratch area) / 24h scratch area × 100%.

[0080] As shown in Figure 7, after culturing the cells in the experimental group with the recombinant collagen solution prepared in Example 1 for 24 hours, the scratch recovery rate was significantly faster than that of the natural collagen treatment group and the blank control group. The scratch area was calculated using ImageJ software, and the results (Figure 8) showed that the scratch recovery area of ​​the LY01 recombinant human collagen treatment group was 17.73%, the natural collagen treatment group was 12.58%, and the PBS treatment group was 4.65%. This indicates that LY01 recombinant human collagen has a migration-promoting activity against BALB / 3T3 cells.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant humanized type III collagen, characterized in that, The amino acid sequence thereof comprises n core units, wherein n is an integer of 1 or greater than 1, and the amino acid sequence of the core unit is shown as SEQ ID NO:

1.

2. The recombinant collagen type III according to claim 1, characterized in that, The n is selected from any one of integers from 4 to 16.

3. The recombinant humanized type III collagen according to claim 1, wherein, The amino acid sequence of the recombinant humanized type III collagen is shown as SEQ ID No.

2.

4. A gene encoding the recombinant humanized type III collagen according to claim 1.

5. A recombinant vector expressing the recombinant humanized type III collagen of claim 1, characterized by, The gene according to claim 4 is included.

6. A recombinant cell expressing the recombinant humanized type III collagen of claim 1, characterized in that, The recombinant cell is transfected with the recombinant vector according to claim 5.

7. The method of producing the recombinant humanized type III collagen according to claim 1, characterized in that, The method comprises the following steps: i) the target gene is assembled on a vector plasmid by cloning, transformed into E. coli for amplification, and the plasmid is extracted; ii) the plasmid is linearized by fast cutting enzyme, and is electrotransformed into yeast, and YPD resistant plates are used for primary screening and secondary screening; iii) the transformants are picked into BMGY culture medium, centrifuged after culture, and the supernatant is discarded; the bacterial body is resuspended using BMMY culture medium, and is induced for culture; the culture solution is separated and purified to obtain the target protein.

8. The method of claim 7, wherein, The vector plasmid is pPICZ alpha A, the E. coli is E. coli JM109, the fast cutting enzyme is SacI enzyme, and the yeast is Pichia pastoris GS115.

9. The method of claim 7, wherein, The primary screening and secondary screening using YPD resistant plates refer to using 100 μg / mL YPD resistant plates for primary screening, and 800 μg / mL YPD resistant plates for secondary screening; the induced culture refers to using 1% methanol, adding once every 24 hours, and inducing culture for 96 hours in total.

10. The recombinant humanized type III collagen according to claim 1, or the recombinant humanized type III collagen prepared by the method according to claim 7, is used in any one of the following applications: i) promoting cell proliferation; ii) promoting cell migration; iii) preparing skin care products and cosmetics; iv) preparing biomedical materials.

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