Recombinant humanized III-type collagen with triple helix structure and application of recombinant humanized III-type collagen

Abstract

The invention provides a recombinant humanized III-type collagen with a triple helix structure and preparation and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant humanized III-type collagen comprises n core units, n is an integer of 1 or greater than 1, and the amino acid sequence of each core unit is as shown in SEQ ID NO: 1; the invention also provides a construction method of the expression system and a preparation method of the recombinant humanized III-type collagen. The invention also provides practical industrial application according to the characteristics of the protein. The recombinant humanized III-type collagen provided by the invention is high in purity, good in stability and high in biological activity, and experiments show that the recombinant humanized III-type collagen has various biological activities of promoting cell proliferation and migration and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to recombinant humanized type III collagen with a triple helix structure and application thereof. Background Art

[0002] Collagen accounts for 25% to 30% of the total protein in the human body. Among the 28 types of collagen, type I, type II, and type III account for 90%. Human type III collagen is a fibrous protein formed by three alpha chain helices. Each protein chain of mature type III collagen contains 1068 amino acids, and the molecular weight of the three protein chains forming a triple helix is ​​close to 400kDa. When used as one of the ingredients of skin care products, the natural micelle structure and high molecular weight characteristics of collagen make it difficult to penetrate the skin barrier to reach the dermis to play a role. Type III collagen contains multiple signal pathway recognition sites, such as binding to integrin recognition sites to activate the regeneration of collagen. The amino acid sequence of type III collagen is a repeating sequence Gly-XY, X and Y can be any amino acids, but X is usually proline (Pro), 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 and cell proliferation activity than single-chain collagen. However, the formation of the triple helix structure of recombinant collagen currently depends 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 first purpose of the present invention is to provide a recombinant humanized type III collagen with a molecular weight smaller than that of natural collagen and a triple helix structure. The present invention obtains its core sequence by screening the mature region sequence of human type III collagen, and obtains a sequence that can self-assemble into a triple helix in vitro through repetition.

[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 conservative amino acid substitution, which allows structurally related amino acids to similarly replace certain amino acids without having a significant impact on the properties of the resulting variant polypeptide, for example, where an amino acid residue is replaced by an amino acid residue with a similar side chain. On the other hand, considering the addition of one or more redundant sequences on the basis of SEQ ID No.1, this change does not bring about a significant increase in biological activity and a more stable structure, that is, the original biologically active fragment will not better exert biological functions such as the ability to bind to receptors due to the intervention of redundant sequences. Therefore, the amino acid changes that fall within the scope of the present invention are achieved by selecting changes that significantly improve the maintenance of biological effects.

[0007] The recombinant humanized type III collagen core sequence of the present invention is repeatedly connected in series 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 series can be a head-to-tail series of core units, or a series of core units connected by a linker, wherein 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 may carry one or more tags composed of histidine. 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 chromatography method using a nickel-based chromatography medium.

[0011] More preferably, the two amino acids Ala and Lys (corresponding to positions 286-287 in the mature region of human type III collagen) are retained at the N-terminus of the recombinant humanized type III collagen, so that the structural stability of the target protein can be further improved.

[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. The sequence is composed of a core sequence (SEQ ID No. 1) repeated 8 times in series, and retains 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_peptide154..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 the protein is not unique. Therefore, the nucleotide sequences that can encode 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 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, cosmids, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), nucleic acid molecules comprising one or more nucleic acid molecules that have been operably 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 promote stable integration of constructs into the host cell genome.

[0018] The present invention provides a recombinant cell expressing the recombinant humanized type III collagen, wherein 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, comprising the following steps:

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

[0022] ii) The plasmid pPICZαA was linearized with the fast-cutting enzyme SacI, electroporated into Pichia pastoris GS115, and primary and secondary screening were performed using YPD resistance plates;

[0023] iii) The transformants are picked up and placed in BMGY medium, and the supernatant is discarded after centrifugation after cultivation; the bacteria are resuspended in BMMY medium and induced to culture; the culture fluid is separated and purified to obtain the target protein.

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

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

[0026] The third object of the present invention is to provide the recombinant type III collagen, and the recombinant type III collagen prepared by the preparation method, including any of the following uses:

[0027] i) Promote cell proliferation;

[0028] ii) promote cell migration;

[0029] iii) preparing cosmetics;

[0030] iv) Preparation of medical materials.

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

[0032] The medical materials include but are not limited to filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug release carriers, wherein 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 protein according to the foregoing 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 prolong 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 ingredients, and can also be mixed with the above-mentioned bioactive ingredients after being prepared separately. The composition also contains additives 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 helical structure in vitro and has good biological activity of promoting cell proliferation and migration. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0041] Figure 6 This is the experimental result of promoting cell proliferation by the recombinant type III collagen LY01 of the present invention.

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

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

[0044] In the present invention, the gene can be synthesized by a biotechnology company. The present invention has no special limitation on the separation and purification method, and conventional protein separation and purification methods in the art can be used. The preferred technical scheme is described in the examples.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, 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 amino acid sequence of the recombinant type III collagen LY01 of the present invention is as follows:

[0048] AKGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERG EPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGERGEPGPRGERGEPGGKGERGAPGEKGEGGPRGDKGETGER(SEQ ID No.2).

[0049] First, a nucleic acid fragment encoding LY01 (SEQ ID No. 3) was synthesized by GenScript Biotech Co., Ltd. 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, electroporated into GS115, and initially screened on 100 ug / mL YPD resistance plates and then rescreened on 800 ug / mL YPD resistance plates.

[0051] Positive transformants were picked into BMGY medium and cultured at 30°C until OD 600 If the cell density is greater than 15, centrifuge and discard the supernatant; resuspend the bacteria in BMMY medium and then induce the culture: use 1% methanol, add it once every 24 hours, and end the culture after a total of 96 hours of induction.

[0052] Separation and purification:

[0053] The fermentation broth was centrifuged at 4000 g to complete solid-liquid separation, and the fermentation supernatant was filtered using a 0.22 μm membrane, followed by overnight dialyzation using a 5 kDa pore size dialysis bag. The dialysis buffer was 20 mM PB buffer at pH 5.0.

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

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

[0056] Buffer A (balanced solution): 20 mM PB pH 5.0,

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

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

[0059] Gradient elution: 0-100% buffer B, collecting the elution peak at 220 nm, 15 CV.

[0060] SDS-PAGE analysis of the purity and expression level of the purified protein showed Figure 1 shown.

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

[0062] The amino acid sequence of the recombinant type III collagen LY02 of the present invention is as follows:

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

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

[0065] The amino acid sequence of the recombinant type III collagen LY03 of the present invention is as follows:

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

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

[0068] The present invention characterizes the structure of collagen by circular dichroism (CD) commonly used in the art. CD is a spectroscopic method used to determine the structure of compounds with chiral structures that can produce left-right optical rotation differential absorption, and is mainly used to determine the asymmetry of molecular structures. Generally, biomacromolecules contain chiral groups and structures, so CD is often used to measure and observe changes in the structure and conformation of biomacromolecules. The CD characteristics of the collagen triple helix structure are generally a positive absorption peak near 221nm and a negative absorption peak near 195nm. The position of the absorption peak will shift with the change of amino acid sequence and length.

[0069] The recombinant collagen solutions prepared in Examples 1 and 2 were subjected to CD testing. The results are shown in Figure 4 and Figure 5 As shown, LY01 and LY02 both have a maximum characteristic positive peak at about 221 nm, and a negative peak appears at less than 200 nm, verifying that the recombinant collagen of the present invention has a triple helix structure.

[0070] Example 5 Experiment on promoting cell proliferation by recombinant type III collagen LY01 of the present invention

[0071] (1) Mouse embryonic fibroblast BALB / 3T3 (Starfish Biotechnology, Cat: TCM-C714) cells were counted and seeded into a 96-well cell culture plate at 2,000 cells / well and placed in a 37° C., 5% CO 2 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 basal culture medium, and 100 μl was added to a cell culture plate. The control group was PBS, and the added volume was 2.5%. The plate was placed in a 37°C, 5% CO2 incubator and cultured for 24 h.

[0073] (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, incubate in a 37°C incubator for 2 hours, and then take out. Use an ELISA reader to read the absorbance value of the 96-well plate at 450nm and record the measurement results (such as Figure 6 ).

[0074] In the cell proliferation experiment, after adding the recombinant collagen solution prepared in Example 1 to the experimental group cells and culturing for 24 hours, the cell proliferation rate was significantly faster than that of the blank control group (PBS), indicating that the recombinant type III collagen LY01 has a proliferation-promoting activity on BALB / 3T3 cells.

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

[0076] The cell scratch test 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) BALB / 3T3 cells were counted and the cells were divided into 2×10 5 Cells / well were inoculated in a 6-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. When the cell density reached 90%, a line was drawn on the 6-well plate with a yellow pipette tip, and the starting scratch position was recorded under a microscope.

[0078] (2) The recombinant collagen solution prepared in Example 1 was diluted to a concentration of 1 μg / ml using basal culture medium, and 2 ml was added to a cell culture plate. The control group was natural collagen, and the blank control group was PBS. The plates were placed in a 37°C, 5% CO2 incubator and cultured for 24 h.

[0079] (3) Take photos under a microscope to record the cells at the same scratch location.

[0080] (4) Image J software was used to calculate the scratch area at the beginning of cell treatment and after 24 hours of treatment, and the wound healing rate was calculated: Wound healing rate = (0 hour scratch area - 24 hour scratch area) / 24 hour scratch area × 100%.

[0081] The results are as follows Figure 7 As shown, after the cells in the experimental group were added with the recombinant collagen solution prepared in Example 1 and cultured for 24 hours, the degree of scratch recovery was significantly faster than that of the natural collagen treatment group and the blank control group. The scratch area was calculated by Image J software, and the results were ( Figure 8) showed that the scratch recovery area of ​​cells in the LY01 recombinant human collagen treatment group was 17.73%, the scratch recovery area of ​​cells in the natural collagen treatment group was 12.58%, and the scratch recovery area of ​​cells in the PBS treatment group was 4.65%. This shows that LY01 recombinant human collagen has the activity of promoting migration of BALB / 3T3 cells.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant humanized type III collagen, characterized in that: The amino acid sequence thereof includes 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 in SEQ ID NO:

1.

2. The recombinant type III collagen according to claim 1, characterized in that The n is selected from any integer between 4 and 16.

3. The recombinant humanized type III collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID No.

2.

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

5. The recombinant vector expressing the recombinant humanized type III collagen according to claim 1, characterized in that: Comprising the gene according to claim 4.

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

7. The method for preparing recombinant humanized type III collagen according to claim 1, characterized in that: The following steps are involved: i) assembling the target gene into a vector plasmid by cloning, transforming it into Escherichia coli for amplification, and extracting the plasmid; ii) Linearize the plasmid with fast-cutting enzyme, electrotransform into yeast, and use YPD resistance plates for primary and secondary screening; iii) The transformants are picked up and placed in BMGY medium, and the supernatant is discarded after centrifugation after cultivation; the bacteria are resuspended in BMMY medium and induced to culture; the culture fluid is separated and purified to obtain the target protein.

8. The method according to claim 7, characterized in that The vector plasmid is pPICZαA, the Escherichia coli is Escherichia coli JM109, the fast-cutting enzyme is enzyme SacI, and the yeast is Pichia pastoris GS115.

9. The method according to claim 7, characterized in that: Using YPD resistance plates for primary screening and rescreening means using 100 μg / mL YPD resistance plates for primary screening and 800 μg / mL YPD resistance plates for rescreening; the induction culture uses 1% methanol, which is added once every 24 hours, for a total of 96 hours of induction culture.

10. The recombinant humanized type III collagen described in claim 1 or the recombinant humanized type III collagen prepared by the preparation method of claim 7 includes any of the following uses: i) Promote cell proliferation; ii) promote cell migration; iii) Preparation of skin care products and cosmetics; iv) Preparation of biomedical materials.

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