A recombinant type A humanized type III collagen and its expression engineering bacteria
A recombinant type A humanized type III collagen produced via Pichia pastoris yeast expression addresses mechanical weakness and production issues, achieving high activity and low cost, suitable for biomedical materials and cosmetics.
Patent Information
- Application Number
- CN202410768271.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-14
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2044-06-14
AI Technical Summary
Existing methods for obtaining collagen, whether from animal tissues or genetic engineering, face issues such as mechanical weakness, cross-contamination risk, complex purification processes, high production costs, and differences in protein activity from natural protein.
A recombinant type A humanized type III collagen is expressed using Pichia pastoris yeast engineered with a specific gene sequence (SEQ ID NO.2) in a pPIC9K vector, followed by purification and separation to achieve high activity and low cost production.
The recombinant collagen has 100% sequence identity to human type III collagen, offering good hydrophilicity and high activity, suitable for biomedical materials and cosmetics, with scalable, simple, and cost-effective production.
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Figure CN118598982B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant type A humanized type III collagen and an expression engineering bacterium thereof. Background Art
[0002] Collagen is a biopolymer material, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for about 30% of the total protein. It has good biocompatibility, low immunogenicity, and tissue degradability and absorbability. It is widely used in wound hemostasis, healing, filling, repair and reconstruction of defective tissues, drug carriers and tissue engineering, and is an ideal medical biomaterial. At present, collagen has been widely used in hemostatic agents and wound dressings, biological patches, bone repair materials, drug release carriers, and as a tissue engineering scaffold. Clinical use shows that collagen has a significant promoting effect on the repair, regeneration and reconstruction of defective tissues.
[0003] At present, there are two main ways to obtain collagen. One is to extract collagen from animal tissues. Although the collagen extracted by this method has good biocompatibility and low degradation, it lacks sufficient mechanical strength and has the risk of cross-contamination. The other method is genetic engineering. The advantages of this method are that the quality of human collagen is controllable, processable, water-soluble, short-term, and low rejection reaction, but it still has disadvantages such as complex purification process, high production cost, and the activity of the expressed protein is very different from that of the natural protein. Summary of the invention
[0004] The purpose of the present invention is to provide a recombinant type A humanized type III collagen and its expression engineering bacteria. The recombinant type A humanized type III collagen provided by the present invention can be expressed by expression engineering bacteria, and the expressed protein has good biological function, low production cost, and is easier to achieve industrialization.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The present invention provides a recombinant type A humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO.1.
[0007] The present invention also provides a gene encoding the protein.
[0008] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.
[0009] The invention also provides an expression vector containing the gene.
[0010] The present invention also provides an expression engineering bacterium, wherein the engineering bacterium contains a gene for expressing recombinant type A humanized type III collagen.
[0011] Preferably, the engineered bacteria contains the gene shown in SEQ ID NO.2.
[0012] The present invention also provides a method for constructing an expression engineering bacterium, comprising the following steps:
[0013] (1) cloning the gene sequence shown in SEQ ID NO.2 into a eukaryotic expression vector to obtain a recombinant plasmid;
[0014] (2) The recombinant plasmid is then transfected into yeast cells to obtain yeast expression engineered bacteria.
[0015] Preferably, the yeast is Pichia pastoris.
[0016] Preferably, the construction method further comprises: culturing and high copy screening the Pichia pastoris expression engineered bacteria in step (2).
[0017] Preferably, the expression vector is pPIC9K.
[0018] The invention provides a method for preparing recombinant type A humanized type III collagen, which comprises using the expression engineering bacteria to express the recombinant type A humanized type III collagen, and then separating and purifying the protein.
[0019] The present invention also provides a use of the above-mentioned recombinant type A humanized type III collagen in the preparation of medicines, cosmetics, foods, health products, and medical devices.
[0020] Beneficial effects:
[0021] The recombinant type A humanized type III collagen gene sequence provided by the present invention is 100% identical to the corresponding part of the human type III collagen gene sequence, and there is no amino acid substitution or mutation. Therefore, it has good hydrophilicity and high activity, making it have application potential in the fields of biomedical materials, beauty cosmetics and skin care products.
[0022] The method for preparing recombinant type A humanized type III collagen provided by the present invention has the advantages of large scale, simplicity, low cost and the like. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0024] Figure 1 Schematic diagram of the construction of recombinant type A humanized type III collagen expression plasmid.
[0025] Figure 2 This is the genome sequencing map of the expression strain COLⅢ-17x3.
[0026] Figure 3 Colony PCR results.
[0027] Figure 4 This is the SDS-PAGE result of the fermentation product of the expression strain COLⅢ-17x3.
[0028] Figure 5 This is the result diagram of recombinant type A human type III collagen freeze-dried powder.
[0029] Figure 6 This is a graph showing the results of a cell proliferation / cytotoxicity experiment using recombinant human type III collagen.
[0030] Figure 7 This is a graph showing the results of recombinant type A human type III collagen promoting cell adhesion. DETAILED DESCRIPTION
[0031] The present invention provides a recombinant type A humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO.1.
[0032] The present invention also provides a gene encoding the above protein. Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.2.
[0033] The present invention provides an expression engineering bacterium, wherein the engineering bacterium contains a gene for expressing recombinant type A humanized type III collagen. Preferably, the engineering bacterium contains the gene shown in SEQ ID NO.2.
[0034] The present invention provides a method for constructing an expression engineering bacterium, comprising the following steps:
[0035] (1) Cloning the gene sequence encoding recombinant type A humanized type III collagen into a eukaryotic expression vector to obtain a recombinant plasmid; the gene sequence is preferably the gene sequence shown in SEQ ID NO.2
[0036] (2) The recombinant plasmid is then transfected into yeast cells to obtain yeast expression engineered bacteria. The yeast is preferably Pichia pastoris.
[0037] Preferably, the construction method further comprises: culturing and high copy screening of the Pichia pastoris expression engineering bacteria in step (2), in order to obtain an expression strain that efficiently secretes and expresses recombinant type A humanized type III collagen.
[0038] The present invention does not impose any particular limitation on the expression vector, but pPIC9K is preferred.
[0039] The base sequences of primers F and R in the present invention are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.
[0040] The present invention provides a method for preparing recombinant type A humanized type III collagen protein, which comprises using the expression engineering bacteria to express the recombinant type A humanized type III collagen protein, and then separating and purifying the protein.
[0041] The present invention also provides an application of the recombinant type A humanized type III collagen in the fields of medicine, cosmetics, food, health products and medical devices.
[0042] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0043] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.
[0044] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.
[0045] Example 1
[0046] Construction of an expression system containing the gene expressing recombinant humanized type III collagen A
[0047] Using pPIC9K (purchased from Invitrogen) as the backbone, the optimized gene sequences were introduced into the multiple cloning sites to obtain pPIC9K-COLⅢ-17x3, which was finally transformed into Pichia pastoris GS115. The specific steps are as follows:
[0048] (1) Based on the mature peptide sequence of human type III collagen (P02461-1) published in the protein resource database UniProt (URL: https: / / www.uniprot.org / ), the highly active sequence was selected and repeated three times to obtain the optimized amino acid sequence of human type III collagen as shown in SEQ ID NO: 1:
[0049] (2) The gene sequence was reversely designed using the online design tool Jcat (http: / / www.jcat.de / ). The Xhol and NotI restriction sites were removed during the design process based on the preferred codons required for expression in the host Pichia pastoris. GenScript Biotech was commissioned to synthesize the optimized COLⅢ-17x3 gene. The optimized gene sequence is shown in SEQ ID NO: 2:
[0050] (3) Construction of recombinant strain: The target gene shown in SEQ ID NO.2 was introduced into the double restriction sites of Xhol and NotI and inserted into the expression vector pPIC9K (containing G418 resistance) to obtain the recombinant plasmid (see Figure 1 ), linearized the recombinant plasmids and electroporated them into Pichia pastoris GS115.
[0051] Example 2
[0052] Screening of Pichia pastoris expressing strains containing recombinant humanized type III collagen A gene
[0053] The recombinants transformed in Example 1 were coated on YPD solid plates containing G418 at concentrations of 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml, respectively, and placed in a 30°C incubator for 2-3 days to observe the growth status of the recombinants. The expression system containing the recombinant human type III collagen expression gene was obtained by colony PCR identification using specific primers F and R. The positive strains could produce bands by PCR, while the negative strains had no bands. The strains were identified by colony PCR and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing (see Figure 2 ), the base sequences of primers F and R are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively. The results of colony PCR are shown in Figure 3 When the PCR result of the colony was as shown in lane 2, the strain was identified as a positive strain.
[0054] Example 3
[0055] Expression of Pichia pastoris containing recombinant humanized type III collagen expression gene The three strains identified positively in Example 2 (labeled 1#, 2#, and 3#) were taken, and 50 μl was inoculated into a conical flask containing 10 ml of BMGY after activation, and cultured overnight at 30°C and 220 r / min, and shaken to OD600 = 2-6 (logarithmic growth, about 16-18 h); centrifuged at 5000 r / min for 5 min at room temperature, collected cells, removed the supernatant, and resuspended the cells with 10 ml of BMMY for induced expression; 1 ml was sampled from the culture medium every 24 h, and methanol was added to a final concentration of 0.5% to continue induction; the samples at the following time points 0, 24, 48, 72, and 96 h were centrifuged at 10000 r / min for 2 min to collect the supernatant, and a 96 h SDS-PAGE run was performed for gel verification (see schematic diagram) Figure 4 , strains 1#, 2#, 3# correspond to Figure 4 Lanes 2-4 in
[0056] Example 4
[0057] Purification of recombinant humanized type III collagen
[0058] The fermentation broth of strain 2# in Example 3 was centrifuged to collect the supernatant, which was filtered through a filter membrane, separated and purified by gel column chromatography, and then freeze-dried to obtain a finished product.
[0059] The specific purification steps of the above human recombinant collagen are as follows: centrifuge the fermentation broth at a speed of 4200rpm for 30min, collect the supernatant, concentrate and wash the supernatant with an ultrafiltration membrane, remove salt and pigment, take an appropriate amount of the above collagen solution, separate and purify it by SP resin column chromatography, collect the eluate containing collagen, wash the eluate with an ultrafiltration membrane, desalt and concentrate it for liquid exchange. Collect the target protein (such as Figure 5 shown).
[0060] Example 5
[0061] Recombinant A-derived type III collagen promotes cell proliferation / cytotoxicity experiment
[0062] BALB / C3T3 cells (mouse embryonic fibroblasts) in logarithmic growth phase were cultured at 1×10 5The cells were inoculated in a 96-well plate at a density of 100 μL per well, divided into a control group and an experimental group. The cells were placed in a carbon dioxide cell culture incubator and cultured for 24 hours at 37°C and 5% CO2. The recombinant type A human type III collagen sample solution obtained in Example 4 was prepared with serum-free culture medium, and the concentrations of the solution were 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, and 2.0 mg / ml, and the solution was sterilized by filtering with a 0.22 μm filter membrane. After 24 hours of conventional culture of BALB / C3T3 cells, the old culture medium was discarded, and 100 μL of serum-free culture medium or 100 μL of recombinant type A human type III collagen sample solution was added. The control group was added with an equal amount of serum-free culture medium, and the experimental group was added with 100 μL of recombinant type A human type III collagen sample solution, with 3 parallel samples in each group. After 24 hours of continuous culture, the culture medium was discarded, and 100 μL of CCK-8 diluted 10 times with serum-free culture medium (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was added to each well, and the cells were placed in a cell culture incubator for another 2 hours. The relative cell proliferation rate was detected using the CCK-8 method, and the absorbance was measured at a wavelength of 450 nm using an enzyme marker. Calculation of cell proliferation rate (RGR) % = absorbance value of the experimental group / absorbance value of the normal control group × 100% (e.g. Figure 6 As shown in the figure, the compound has a certain cell proliferation ability and no cytotoxicity within the selected concentration range, and the preferred concentration is 0.1 mg / ml to 1.0 mg / ml.
[0063] Example 6
[0064] Comparative experiment on adhesion of recombinant type A human type III collagen
[0065] Competitors 1 and 2 (type III collagen products from different companies) and the patented recombinant type A human type III collagen were prepared at 0.5 mg / ml and coated on a 96-well plate. 3w BALB / C3T3 cells were added to each well at 4°C for 24 hours and cultured with DMEM medium. After 1 hour, the adhesion was observed, the supernatant was removed, and the cells were washed twice with PBS. PBS was added to each well, and the cck8 method was used. The OD was measured at a wavelength of 450 nm using an enzyme marker to obtain the relative cell adhesion rate (see the schematic diagram). Figure 7 ). The cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better the external environment it can provide to the cells in a short time, helping the cells to adhere to the wall. The cell adhesion rate can reflect the activity of collagen. With the adhesion rate of the blank group as 1, the relative cell adhesion activity of competitors 1, 2 and recombinant type A human type III collagen can be measured.
[0066] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A recombinant type A humanized type III collagen, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.
1.
2. A gene encoding the recombinant type A humanized type III collagen according to claim 1.
3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.
2.
4. An expression vector containing the gene according to claim 2 or 3.
5. An expression engineering bacterium, characterized in that: The engineered bacteria contains a gene for expressing the recombinant type A humanized type III collagen according to claim 1.
6. The engineered bacteria according to claim 5, characterized in that The engineered bacteria contains the nucleotide sequence shown in SEQ ID NO.
2.
7. A method for constructing an expression engineering bacterium, characterized in that: The following steps are involved: (1) cloning the gene sequence shown in SEQ ID NO.2 into a eukaryotic expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid is then transfected into yeast cells to obtain yeast expression engineered bacteria.
8. The method for constructing an expression engineering bacterium according to claim 7, characterized in that: The eukaryotic expression vector is pPIC9K.
9. A method for preparing recombinant type A humanized type III collagen, characterized in that: The expression engineering bacteria described in claim 5 are used to express recombinant type A humanized type III collagen, and then separated and purified.