Recombinant Humanized Type III Collagen with a Triple-Helical Structure and Its Applications

Abstract

The present invention provides a recombinant humanized type III collagen having a triple helix structure, its preparation method and applications, belonging to the technical field of protein engineering. The amino acid sequence of the recombinant humanized type III collagen comprises n core units, where n is an integer of 1 or greater than 1, and the amino acid sequence of the core unit is as shown in SEQ ID NO: 1. The present invention also provides a method for constructing an expression system and a method for preparing the recombinant humanized type III collagen. According to the characteristics of this protein, the present invention also provides practical industrial applications. The recombinant humanized type III collagen provided by the present invention has high purity, good stability and high biological activity. Experiments show that it has various biological activities such as promoting cell proliferation and migration.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to recombinant humanized type III collagen with a triple helix structure and its applications. Background Art

[0002] Collagen accounts for 25% - 30% of the total human body protein, and types I, II, and III among 28 types of collagen account for 90%. Human type III collagen is a fibrin formed by the helix of three alpha chains. Each protein chain of mature type III collagen contains 1068 amino acids, and the three protein chains form a triple helix with a molecular weight close to 400 kDa. When used as one of the components of skin care products, the natural micelle structure and large molecular weight characteristics of collagen make it difficult to penetrate the skin barrier and reach the dermis to play a role. There are multiple signal pathway recognition sites on type III collagen, such as binding to integrin recognition sites to activate the regeneration of collagen. The amino acid sequence of type III collagen is a repetitive sequence Gly-X-Y, where X and Y can be any amino acids, but X is usually proline (Pro), and Y is usually hydroxyproline (Hyp) and hydroxylysine (Hyl), and Hyp and Hyl need to be modified by hydroxylase.

[0003] It is reported that collagen with a triple helix structure has better stability, biocompatibility, cell proliferation activity, etc. than single-chain collagen. However, at present, the formation of the triple helix structure of recombinant collagen mainly depends on the matching between the sequence and hydroxylase from different sources, and there are few reports on recombinant collagen that can self-assemble into a triple helix. Summary of the Invention

[0004] The first object of the present invention is to provide a recombinant humanized type III collagen with a molecular weight smaller than that of natural collagen and having a triple helix structure. The present invention screens the mature region sequence of human type III collagen to obtain its core sequence, and through repetition, obtains a sequence that can self-assemble into a triple helix in vitro.

[0005] The amino acid sequence of the core sequence of the recombinant humanized type III collagen of the present invention has a homology of more than 90% 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%, 100%; preferably, the amino acid sequence of the core sequence of the recombinant humanized type III collagen is as shown in SEQ ID No.1.

[0006] Regarding amino acid sequence homology, on the one hand, it is based on conservative amino acid substitutions. This conservatism enables structurally related amino acids to similarly substitute a certain amino acid without significantly affecting the properties of the resulting variant polypeptide. For example, an amino acid residue is substituted by an amino acid residue with a similar side chain. On the other hand, considering the addition of one or more redundant sequences based on SEQ ID No.1, and this change does not bring about a significant increase in biological activity or a more stable structure, that is, the original bioactive fragment will not better exert its biological functions such as the ability to bind to receptors due to the intervention of redundant sequences. Therefore, amino acid changes falling within the scope of the present invention are achieved by selecting those changes that significantly enhance the maintenance of biological effects.

[0007] The core sequence of the recombinant humanized type III collagen described in the present invention is repeatedly concatenated to form a sequence that can self-assemble into a triple helix in vitro.

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

[0009] The repeated concatenation can be the head-to-tail concatenation of core units, or the core units can be concatenated by a linker. The linker preferably contains glycine (Gly) and / or serine (Ser), and the linker preferably contains 1 - 5 amino acids.

[0010] The recombinant collagen of the present invention can carry a tag composed of one or more histidines. The tag is connected to the N-terminus or C-terminus of the recombinant collagen. The tag can be used to purify the protein by a chromatographic method using a nickel-based chromatographic medium.

[0011] More preferably, it is obtained by retaining two amino acids, Ala and Lys, at the N-terminus of the recombinant humanized type III collagen (corresponding to positions 286 - 287 of the mature region of human type III collagen), 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 as shown in SEQ ID No.2. This sequence is formed by repeatedly concatenating the core sequence (SEQ ID No.1) 8 times and retaining two amino acids, Ala and Lys, at the N-terminus.

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

[0014] The present invention provides a gene encoding the recombinant humanized type III collagen, and the nucleotide sequence of the gene has a homology of more than 80% with the nucleotide sequence of SEQ ID No. 3; preferably, the nucleotide sequence of the gene has a homology of more than 90% 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, the nucleotide sequences capable of encoding the amino acid sequence of the recombinant humanized type III collagen of the present invention are all within the protection scope of the present invention.

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

[0017] A vector includes any nucleic acid molecule (such as a plasmid, cosmid, virus, self-replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule) derived from any source and capable of genomic integration or autonomous replication, which contains a nucleic acid molecule having one or more nucleic acid molecules operably linked. A vector may include, for example, one or more selectable markers, one or more origins of replication (such as prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that promote stable integration of the construct into the host cell genome.

[0018] The present invention provides a recombinant cell for expressing the recombinant humanized type III collagen, and the recombinant vector is transfected into the recombinant cell.

[0019] The recombinant cell is preferably Pichia pastoris GS115.

[0020] The second object of the present invention is to provide a method for expressing the recombinant humanized type III collagen, preferably a method for expressing in Pichia pastoris GS115, including the following steps:

[0021] i) Assembling the target fragment onto a vector plasmid by cloning, transforming it into Escherichia coli JM109 for amplification, and extracting the plasmid;

[0022] ii) Linearizing the plasmid pPICZαA with the fast-cutting enzyme SacI, electrotransforming it into Pichia pastoris GS115, and performing primary screening and rescreening using a YPD resistant plate;

[0023] iii) Picking the transformants into BMGY medium, culturing, centrifuging, and discarding the supernatant; resuspending the cell pellet with BMMY medium and inducing culture; the culture solution is separated and purified to obtain the target protein.

[0024] Preferably, the primary screening and rescreening using YPD resistance plates refers to primary screening using YPD resistance plates with a concentration of 100 μg / mL and rescreening using YPD resistance plates with a concentration of 800 μg / mL.

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

[0026] The third object of the present invention is to provide the following applications of the recombinant type III collagen and the recombinant type III collagen obtained by the preparation method:

[0027] i) Promote cell proliferation;

[0028] ii) Promote cell migration;

[0029] iii) Prepare cosmetics;

[0030] iv) Prepare medical materials.

[0031] The cosmetics include but are not limited to skin care lotions, skin care creams, essence waters, facial masks, scalp serums, and shampoos.

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

[0033] The present invention also provides a composition comprising a cosmetically or pharmaceutically effective amount of at least one of the aforementioned proteins 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; further, in order to promote the transdermal absorption of collagen molecules, solid carriers such as non-woven fabrics can be used to prepare patch products, which are applied to the face to extend the action time of the collagen solution on the skin surface. The recombinant type III collagen of the present invention can be prepared and used in series with recombinant or natural type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type XVII collagen, fibronectin, human epidermal growth factor and other bioactive components, and can also be mixed with the above bioactive components after separate preparation. The composition also contains additive components such as stabilizers, preservatives, and carriers. The additives include but are not limited to liposomes, biodegradable microcapsules, aerosols, powders, glycerol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, and tocopherol.

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

[0035] The recombinant type III collagen of the present invention can self-assemble into a stable triple helix structure in vitro and has good bioactivity in promoting cell proliferation and migration. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0039] Figure 4 It is the circular dichroism (CD) diagram of the recombinant type III collagen LY01 of the present invention.

[0040] Figure 5 It is the circular dichroism (CD) diagram of the recombinant type III collagen LY02 of the present invention.

[0041] Figure 6 It is the result of the cell proliferation promotion experiment of the recombinant type III collagen LY01 of the present invention.

[0042] Figure 7 It is the result of the cell scratch experiment of the recombinant type III collagen LY01 of the present invention.

[0043] Figure 8 It is the confluence of the cell scratch experiment of the recombinant type III collagen LY01 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0044] In the present invention, the gene can be obtained by synthesis from a biotechnology company. The present invention has no special limitation on the method of separation and purification, and conventional protein separation and purification methods in the art can be used. Preferred technical solutions are described in the examples.

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

[0046] Example 1 SDS-PAGE Experiment of the Recombinant Type III Collagen LY01 of the Present Invention

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

[0048] AKGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGER(SEQ ID No.2).

[0049] First, the nucleic acid fragment encoding LY01 (SEQ ID No.3) was synthesized by GenScript Biotech Corporation. The target fragment was assembled into the vector plasmid pPICZαA by one-step cloning, transformed into Escherichia coli JM109, amplified, and the plasmid was extracted.

[0050] The plasmid was linearized with the fast-cutting enzyme SacI and electrotransformed into GS115. Preliminary screening was carried out using a YPD resistance plate of 100 μg / mL, and rescreening was carried out using a YPD resistance plate of 800 μg / mL.

[0051] The positive transformants were picked into BMGY medium and cultured at 30 °C until the OD 600 was greater than 1.5. The supernatant was discarded by centrifugation; the cell pellet was resuspended with BMMY medium and then induced for culture: 1% methanol was used and replenished every 24 hours, and the culture was ended after a total of 96 hours of induction.

[0052] Isolation and purification:

[0053] The fermentation broth was subjected to solid-liquid separation at a centrifugal force of 4000g. The fermentation supernatant was filtered through a 0.22 μm membrane, and then dialyzed overnight using a dialysis bag with a pore size of 5 kDa. The dialysis buffer was 20 mM PB buffer at pH 5.0.

[0054] Then, elution was carried out using a chromatography column HiTrap SP HP 5ml (Cytiva), and the parameters were set as follows:

[0055] Sample environment: 20 mM PB pH 5.0;

[0056] Buffer A (equilibration buffer): 20 mM PB pH 5.0,

[0057] Buffer B (eluent): 20 mM PB pH 5.0, 1 M NaCl;

[0058] Experimental flow rate: 4 ml / min;

[0059] Gradient elution: Elute with 0 - 100% Buffer B, collect the elution peak at 220 nm, 15 CV.

[0060] Analyze the purity and expression level of the purified protein by SDS - PAGE, and the results are as Figure 1 shown.

[0061] Example 2 SDS - PAGE experiment of the recombinant type III collagen LY02 of the present invention

[0062] The recombinant type III collagen LY02 of the present invention has the following specific amino acid sequence:

[0063] AKGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGER(SEQ ID No.4). The nucleic acid fragment encoding LY02 was synthesized by GenScript. Other steps refer to Example 1. The results of SDS-PAGE analysis of the purified protein are as Figure 2 shown.

[0064] Example 3 SDS-PAGE experiment of recombinant type III collagen LY03 of the present invention

[0065] The recombinant type III collagen LY03 of the present invention has the following specific amino acid sequence:

[0066] AKGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGER (SEQ ID No.5). The nucleic acid fragment encoding LY03 was synthesized by GenScript. Other steps refer to Example 1. The results of SDS-PAGE analysis of the purified protein are as Figure 3 shown.

[0067] Example 4 Structural Characterization of the Recombinant Type III Collagen of the Present Invention

[0068] The structural characterization of the collagen of the present invention was performed using circular dichroism (CD), a commonly used method in the art. CD is used to determine the structure of compounds with chiral structures that can produce differential absorption of left and right circularly polarized light, and is mainly a spectroscopic method for determining the molecular structure asymmetry. Generally, biological macromolecules contain chiral groups and structures, so CD is often used to measure and observe the structural and conformational changes of biological macromolecules. The CD characteristics of the collagen triple helix structure generally have a positive absorption peak near 221 nm and a negative absorption peak near 195 nm, and the positions of the absorption peaks will shift with changes in the amino acid sequence and length.

[0069] The recombinant collagen solutions prepared in Examples 1 and 2 were subjected to CD detection. The results are as Figure 4 and Figure 5 shown. Both LY01 and LY02 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.

[0070] Example 5 Cell Proliferation Promotion Experiment of the Recombinant Type III Collagen LY01 of the Present Invention

[0071] (1) After cell counting of mouse embryonic fibroblasts BALB / 3T3 (Haixing Bio, Cat: TCM-C714), they were seeded into a 96-well cell culture plate at 2000 cells / well and incubated statically in a 37 °C, 5% CO2 cell culture incubator for 24 h.

[0072] (2) The recombinant collagen solution prepared in Example 1 was diluted to a concentration of 1 μg / ml using the basal medium, and 100 μl was taken and added to the cell culture plate. The control group was PBS, and the added volume was 2.5%. It was further incubated in a 37 °C, 5% CO2 incubator for 24 h.

[0073] (3) Prepare the CCK8 working solution. Dilute the CCK8 reagent with DMEM medium at a ratio of 1:10. Remove the supernatant, add the prepared CCK8 working solution, and incubate it in an incubator at 37 °C for 2 hours and then take it out. Read the absorbance value of the 96-well plate at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and record the measurement results (such as Figure 6 ).

[0074] After culturing the experimental group cells in the cell proliferation experiment 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 a proliferative activity promoting effect on BALB / 3T3 cells.

[0075] Example 6 Cell scratch assay of the recombinant type III collagen LY01 of the present invention

[0076] The cell scratch assay is a commonly used method to detect the cell migration activity of collagen. The higher the migration rate, the better the biological activity of collagen. The specific steps are as follows:

[0077] (1) After counting the BALB / 3T3 cells, inoculate them into a 6-well cell culture plate at 2×10 5 cells / well and culture them in an incubator at 37 °C with 5% CO2. When the cell density reaches 90%, draw a line on the 6-well plate with a yellow pipette tip and take a photo under the microscope to record the starting scratch position.

[0078] (2) Dilute the recombinant collagen solution prepared in Example 1 with a basal medium to a concentration of 1 μg / ml, take 2 ml and add it to the cell culture plate. The control group is natural collagen, and the blank control group is PBS. Continue to culture it in an incubator at 37 °C with 5% CO2 for 24 h.

[0079] (3) Take a photo of the cells at the same scratch position under the microscope.

[0080] (4) Use Image J software to calculate the scratch area at the beginning and after 24 h of treatment of the cells, and calculate the wound healing ratio. Wound healing ratio = (scratch area at 0 h - scratch area at 24 h) / scratch area at 24 h × 100%.

[0081] The results are as Figure 7 shown. After culturing the experimental group cells with the recombinant collagen solution prepared in Example 1 for 24 h, the degree of scratch recovery was significantly faster than that of the natural collagen treatment group and the blank control group. Calculate the scratch area through Image J software, and the results ( Figure 8)It shows that the cell scratch recovery area of the LY01 recombinant human collagen treatment group is 17.73%, the cell scratch recovery area of the natural collagen treatment group is 12.58%, and the cell scratch recovery area of the PBS treatment group is 4.65%. It shows that LY01 recombinant human collagen has a pro-migration activity on BALB / 3T3 cells.

[0082] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant humanized type III collagen, characterized in that, The amino acid sequence of the recombinant humanized type III collagen is as shown in SEQ ID No.2; wherein, the amino acid sequence includes 8 core units, and the amino acid sequence of the core unit is as shown in SEQ ID NO:

1.

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

3. A recombinant vector expressing recombinant humanized type III collagen, characterized in that, Comprising the gene according to claim 2.

4. A recombinant cell expressing recombinant humanized type III collagen, characterized in that, The recombinant vector according to claim 3 is transfected into the recombinant cell.

5. The preparation method of the recombinant humanized type III collagen according to claim 1, characterized in that, Comprising the following steps: i) The target gene is cloned and assembled onto the vector plasmid, transformed into Escherichia coli for amplification, and the plasmid is extracted. ii) The plasmid is linearized with a fast-cutting enzyme, electrotransformed into yeast, and preliminarily screened and rescreened using a YPD resistance plate. iii) The transformant is picked into the BMGY medium, cultured and centrifuged to discard the supernatant; the cell pellet is resuspended with the BMMY medium and induced to culture; the culture solution is separated and purified to obtain the target protein.

6. The method according to claim 5, characterized in that, The vector plasmid is pPICZαA, the Escherichia coli is Escherichia coli JM109, the fast-cutting enzyme is the enzyme SacI, and the yeast is Pichia pastoris GS115.

7. The method according to claim 5, characterized in that, Using the YPD resistance plate for primary screening and rescreening means using a 100 μg / mL YPD resistance plate for primary screening and an 800 μg / mL YPD resistance plate for rescreening; the induced culture is carried out using 1% methanol, supplemented once every 24 hours, for a total of 96 hours of induced culture.

8. Use of the recombinant humanized type III collagen according to claim 1 and the recombinant humanized type III collagen prepared by the preparation method according to claim 6 in promoting cell proliferation or promoting cell migration, and the use is for non-therapeutic purposes.

9. Use of the recombinant humanized type III collagen according to claim 1 and the recombinant humanized type III collagen prepared by the preparation method according to claim 6 in the preparation of skin care products, cosmetics or biomedical materials.

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