High-expression recombinant collagen allosteric body, construction method and application of high-expression recombinant collagen allosteric body

Abstract

The invention discloses a high-expression recombinant collagen allosteric body as well as a construction method and application thereof, a recombinant gene is formed by recombining a type III collagen gene, and stable GXY triad mutation is performed on the basis of an original collagen sequence. The sequence of the recombinant protein is shown as SEQ ID NO.2, the recombinant collagen obtained by the method has similar physicochemical properties and biological functions with the original sequence, and can be applied to injection filling type anti-aging wrinkle removal, facial rejuvenation, skin beautifying, nursing and anti-aging and patch type skin repair dressings, and the active collagen has the fields of repair, moisturizing, whitening and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of humanized collagen, and more specifically to a highly expressed recombinant collagen variant, a construction method and application thereof. Background Art

[0002] Human collagen is the main structural protein in the extracellular matrix and plays a variety of important biological functions. Recombinant collagen is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies, with the gene encoding of the functional region of a specific type of human collagen as a template for screening and preparation to obtain the same or similar amino acid sequence as human collagen. Among them, recombinant humanized collagen that is consistent with the full-length or partial amino acid sequence of human collagen does not contain sequence fragments of non-human collagen, has a low risk of immunogenicity, and has original intellectual property rights in my country. At present, recombinant humanized collagen is gradually being promoted and applied in the biomedical field.

[0003] At present, collagen is still mainly extracted from animal tissues or human corpses / placentas. Collagen from xenogeneic and allogeneic sources has the following significant problems: pathogen residues, strong acid and alkali residues, low biological activity, low water solubility, viral contamination and immune rejection. It is not suitable for use as a biomaterial and is difficult to apply in the medical field.

[0004] Therefore, how to provide a recombinant humanized collagen that is free of viral hazards, non-immunogenic, and highly hydrophilic is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a recombinant humanized collagen.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] One aspect of an embodiment of the present invention provides a gene, the nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0008] A second aspect of an embodiment of the present invention provides a recombinant humanized collagen protein, wherein the recombinant humanized collagen protein is encoded by the sequence shown in SEQ ID NO.1.

[0009] The third aspect of the embodiments of the present invention provides a method for constructing a highly expressed recombinant humanized collagen variant, wherein a stable GXY triplet mutation is performed on the original collagen coding sequence, and the gene sequence after the mutation is shown in SEQ ID NO.1.

[0010] In a preferred embodiment, G in the GXY triplet is glycine, X and Y can be any amino acids, and the mutation stabilizing the GXY triplet is a GER-GAR mutation.

[0011] A fourth aspect of the embodiments of the present invention provides the use of recombinant humanized collagen in the preparation of wound dressings.

[0012] Preferably, the skin tissue preparation is used to maintain or promote the proliferation of human skin fibroblasts

[0013] From the above technical solutions, we can know that compared with the existing technology, the recombinant humanized collagen we produce is the collagen produced by transferring the human collagen gene into the engineered bacteria / engineered cells for expression after specific enzyme cleavage and splicing. Compared with animal-derived collagen, it has the following outstanding advantages:

[0014] 1) Strong biological activity and processability. The recombinant humanized collagen prepared by the present invention can form a triple-repeating helical structure, which not only enables it to have biological activity under natural conditions, but also has processability under the condition that the molecular weight and physicochemical properties remain unchanged. Animal-derived collagen has a triple-cross helical structure, and the molecules are easily broken during the processing process, causing changes in the physicochemical properties.

[0015] 2) No virus risk and no immunogenicity make recombinant human collagen more widely used in the field of medical engineering

[0016] 3) Through genetic design, recombinant collagen can be made more hydrophilic / water-soluble than natural molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The attached figure is a structural diagram of the recombinant gene sequence.

[0019] Figure 2 The attached figure is a diagram of plasmid linearization results; M: DL5000 DNA Marker; 1: before linearization; 2: after linearization.

[0020] Figure 3 The attached figure is a graph of the electrophoresis results of the PCR reaction; M: DL5000 DNA Marker; 1-20: PCR results of different clones; -: negative control (sterile water); +: positive control.

[0021] Figure 4 The attached figure is a PCR result diagram; M: Protein marker; 1-10: colonies that are positive in PCR detection;

[0022] Figure 5 The attached figure is a graph showing the proliferation results of mouse fibroblasts. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The materials and reagents used in the examples are:

[0025]

[0026]

[0027] Example 1 Construction of recombinant expression vector

[0028] This sequence uses the 486-879 amino acids in the type III collagen sequence (https: / / www.uniprot.org / uniprotkb / P02461 / entry) as monomers, and the sequence is as shown in SEQ ID NO.1:

[0029] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGE RGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARG.

[0030] The unstable GER in the GXY triplet was mutated to GAR, and its sequence is shown in SEQ ID NO.2:

[0031] GAPGFRGPAGPNGIPGEKGPAGARGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGA RGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGARGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGARGETGPPGPAGFPGAPGQNGEPGGKGARGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGARGSPGGPGAAGFPGARG.

[0032] The amino acid sequence was optimized for Pichia pastoris codons to obtain its corresponding gene sequence, which is shown in SEQ ID NO.3:

[0033]

[0034] We commissioned Global Genetics to synthesize the DNA fragment shown in SEQ ID NO.3, and then fused the DNA fragment into the pPICZ(alpha)A plasmid after double digestion with XhoI and SalI. Figure 1 ) to obtain a recombinant expression vector expressing the protein sequence of SEQ ID NO.2.

[0035] Example 2 Construction of recombinant engineering strains, induced expression and strain screening

[0036] (1) Linearization of recombinant plasmid

[0037] Select the appropriate linearization site SacI for the constructed expression plasmid and perform single enzyme digestion according to the following system:

[0038] Enzyme digestion system (40 μl):

[0039] 4μl FD Buffer (enzyme digestion buffer)

[0040] 3 μl FastDigest TM SacI endonuclease

[0041] 33 μl plasmid

[0042] After incubation in a 37°C water bath for 1 hour, samples were taken and run to verify the linearization effect ( Figure 2 ).

[0043] (2) Linearized plasmid transformation into expression strain

[0044] Add 10 μl of the linearized plasmid to 100 μl of the X33 competent cells prepared in advance, mix well, transfer to a pre-cooled electroporation cup (2 mm), and perform electroporation. The electroporator is set to 1.8 kV, and the duration is 4.5 ms to 5 ms. Immediately after the electroporation, add 300 μl of 1 M sorbitol, and then add 200 μl of YPD medium. After 3 hours of recovery at 30 ° C, spread on a YPD plate containing Zeocin antibiotics and culture at 30 ° C for 2 to 3 days until a single colony appears.

[0045] (3) Identification of positive clones by colony PCR

[0046] Dissolve the single clones on the plate with 10 μl ddH2O, take 0.5 μl each as a template, and use the corresponding universal primers for PCR identification ( Figure 3 ).

[0047] Primer sequences:

[0048] 5ˋAOX1: GACTGGTTCCAATTGACAAGC, shown in SEQ ID NO.4;

[0049] 3ˋAOX1: CGTTACCCTTTGGACCTGGA, as shown in SEQ ID NO. 5. PCR system (10 μl): 5 μl mix enzyme, 0.2 μl primer F, 0.2 μl primer R, 0.5 μl template, 4.1 μl ddH2O.

[0050] PCR program: 95°C for 10 min, 95°C for 30 s, 55°C for 30 s (30 cycles), 72°C for 1 min / kb, 72°C for 5 min.

[0051] Example 3 Protein Expression Identification

[0052] (1) Selection and cultivation of positive clones

[0053] Select a small amount of the better positive clone bacterial solution and transfer it to a 24-well culture plate containing 2 ml of BMGY medium, and culture it at 30°C, 200r overnight.

[0054] (2) Inducible expression

[0055] The next day, take out the cultured 24-well plate and let it stand outside for 30 minutes, then carefully aspirate the supernatant, add 3 ml of BMMY medium to the bacterial solution, and add methanol (usually 1 / 100), and then induce expression at 30°C, 200r. After each 24h of culture, add an equal amount of methanol until the induced expression is completed (inducible expression for 3 days).

[0056] (3) Collection and treatment of bacterial culture

[0057] After the induction expression was completed, the bacterial solution was collected into a 1.5 ml EP tube, centrifuged at 10000 rpm for 1 min, the culture medium was separated, 100 ul of the supernatant was taken, 25 ul of 5× reducing loading buffer was added, and boiled for 10 min.

[0058] (4) SDS-PAGE analysis

[0059] The treated samples were subjected to SDS-PAGE electrophoresis to compare the protein band intensities of the mutant and wild type to evaluate the changes in protein expression after mutation ( Figure 4 ).

[0060] Example 4 The efficacy of the recombinant humanized type III collagen described in Examples 1-3 in stimulating the proliferation of mouse skin fibroblasts.

[0061] Mouse fibroblasts (purchased from Pricella) were cultured according to conventional cell biology methods.

[0062] (1) Mouse fibroblasts (NIH 3T3) were cultured in DMEM medium containing 10% FBS. When the cells grew to 80-90% confluence, they were digested with trypsin-EDTA and resuspended in fresh medium.

[0063] (2) Adjust the cell suspension to an appropriate cell concentration (1×10^4 cells / well) and evenly inoculate into a 96-well culture plate, adding 100 μl of cell suspension to each well. Place the culture plate in a cell culture incubator and incubate for 24 hours to allow the cells to adhere and recover.

[0064] (3) Control group: add 100 μl of DMEM medium containing 10% FBS. Experimental group: add 100 μl of DMEM medium containing 10% FBS and different concentrations of recombinant collagen (0, 200, 500, 1000, 2000, 5000 μg / ml).

[0065] (4) After 48 hours of treatment, use a cell counting kit (CCK-8 kit) to detect cell proliferation. Add 10 μl of CCK-8 reagent to each well and continue incubation for 2 hours. Use an enzyme reader to measure the absorbance (OD value) of each well at 450 nm.

[0066] (5) Experimental results

[0067] Table 1 Experimental results of promoting mouse fibroblast proliferation

[0068] Group Hole 1 Hole 2 Hole 3 Hole 4 Hole 5 Hole 6 Control group 0.200 0.195 0.205 0.198 0.196 0.202 Sample set 0.196 0.225 0.268 0.317 0.418 0.396

[0069] The results are shown in Table 1. Figure 5 As shown in the figure, after adding different concentrations of recombinant collagen to the experimental groups, the proliferation of mouse fibroblasts was significantly different. The cell proliferation was significantly increased in the higher concentration recombinant collagen groups (50, 100, 200, 500, 1000, 2000, 5000 μg / ml). After 48 hours of treatment, the cell proliferation rate was significantly increased. ( Figure 5 )

[0070] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A gene, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. Recombinant humanized collagen, characterized in that: The recombinant humanized collagen is encoded by the sequence shown in SEQ ID NO.

3.

3. A method for constructing a highly expressed recombinant humanized collagen variant, characterized in that: A stable GXY triplet mutation was performed on the original collagen coding sequence, and the gene sequence after the mutation is shown in SEQ ID NO.

3.

4. The method according to claim 3, characterized in that In the GXY triplet, G is glycine, X and Y can be any amino acids, and the mutation stabilizing the GXY triplet is a GER-GAR mutation.

5. The use of the recombinant humanized collagen according to claim 2 in the preparation of a product for skin tissue wounds, characterized in that: The skin tissue preparation is used for maintaining or promoting the proliferation of human skin fibroblasts.

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