A recombinant type I collagen α1 chain polypeptide and gene, vector, bacteria and application

Abstract

The present invention provides a recombinant type I collagen α1 chain polypeptide, the amino acid sequence of which is shown in SEQ ID NO.2, and contains two "GER integrin recognition sites". The present invention also provides a recombinant type I collagen α1 chain polypeptide encoding gene, as shown in SEQ ID NO.1, for encoding the above-mentioned recombinant type I collagen α1 chain polypeptide. At the same time, the present invention also provides a recombinant expression vector containing the above-mentioned recombinant type I collagen α1 chain polypeptide encoding gene, a recombinant Pichia pastoris engineering bacterium, and a preparation method and application of the above-mentioned recombinant type I collagen α1 chain polypeptide. The recombinant type I collagen α1 chain polypeptide designed by the present invention has a small molecular weight, contains two GER integrin recognition sites, has excellent activity, has a certain migration effect in human immortalized epidermal cells, and can be widely used in medical cosmetology, cosmetics and other fields.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant type I collagen alpha 1 chain polypeptide and its gene, vector, bacteria and application. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, and is widely used in medical materials, cosmetics, and food industries.

[0003] At present, collagen is mainly extracted from animal connective tissue by acid and alkali hydrolysis, but the resulting product is complex in composition and has risks such as animal-derived diseases. At the same time, large-scale preparation puts tremendous pressure on animal husbandry on the supply side. With the development of science and technology, many researchers use genetic engineering methods to encode human collagen genes in host cells, and use modern gene recombination technology to express the target protein in host cells, thereby obtaining recombinant human collagen. Recombinant human collagen products have many advantages, such as high safety, stable quality, advanced technology, no pollution, low cost, and large-scale production, which solves many defects of traditional extraction methods and increases economic and social benefits.

[0004] Type I human collagen is a major protein component of the extracellular matrix in bones, skin and tendons. It is mainly secreted by osteoblasts, dermal fibroblasts and tenocytes, and is mainly composed of 2 α1(I) chains and 1 α2(I). Mutations in the gene encoding type I collagen can cause osteogenesis imperfecta, Ehlers-Danlos syndrome, etc. In the skin, type I collagen is the main component of the dermis, which mainly maintains skin tension and withstands tension, providing a strong support structure and support for the skin. The loss of type I collagen can cause facial wrinkles and depressions, so the purpose of moisturizing and removing wrinkles can be achieved by supplementing collagen to the skin. As the most common collagen in the human body, type I collagen has the characteristics of wide distribution and high content compared to other types of collagen. This feature determines that it has a wider application in tendons, bones and other fields than other types of collagen. For this reason, the production of type I collagen is of great significance to my country's medical health and beauty industry.

[0005] With the rapid development of genetic engineering technology, many recombinant expression systems have been applied to the recombinant expression of human collagen, among which microbial expression systems are the most widely used. Yang Xia et al. used Escherichia coli to express human collagen type I peptide (Chinese Patent No. ZL202110968550.2), and Xiao Jianxi et al. expressed a type I collagen through Escherichia coli (Chinese Patent No. ZL202210630810.X), but the Escherichia coli expression system still faces some difficult problems. The collagen produced has endotoxins, the purification steps are complicated, and there is a lack of post-translational modification. Pichia pastoris can secrete and express recombinant human collagen, and the collagen produced does not contain endotoxins and has a simple purification process. Therefore, it is possible to try to construct a human type I collagen expression system based on Pichia pastoris.

[0006] At the same time, integrin is a transmembrane receptor, a heterodimer formed by two subunits, α and β, which can mediate signal transmission in the intracellular and extracellular environment. Therefore, integrins not only have the mechanical effect of passing through the membrane, but also have the ability to participate in cell messaging, cell cycle regulation, cell morphology and cell movement. The amino acid sequence of the integrin recognition site includes GER, etc. Collagen fragments containing the corresponding amino acid sequence and with the site exposed to the outside can be recognized by the integrins on the cell membrane and promote cell-related functions through signal transduction. Therefore, on the basis of human type I collagen, through sequence design and optimization based on integrin recognition sites, recombinant type I collagen with better biological activity can be obtained. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant type I collagen α1 chain polypeptide and gene, vector, bacteria and application. The obtained recombinant type I collagen α1 chain polypeptide has a small molecular weight and excellent activity. It has a certain migration promoting effect in human immortalized epidermal cells and can be widely used in medical cosmetology, cosmetics and other fields.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A recombinant type I collagen α1 chain polypeptide, the amino acid sequence of the recombinant type I collagen α1 chain polypeptide is shown as SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the recombinant type I collagen α1 chain polypeptide contains two "GER integrin recognition sites".

[0011] A recombinant type I collagen α1 chain polypeptide encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.1, is used to encode the above recombinant type I collagen α1 chain polypeptide.

[0012] As one of the preferred embodiments of the present invention, the nucleotide sequence shown in SEQ ID NO.1 is a sequence after codon optimization for the codon preference of Pichia pastoris, wherein the corresponding original recombinant human type I collagen codon is optimized to a codon suitable for Pichia pastoris using online codon optimization software without changing the protein coding sequence.

[0013] A recombinant expression vector contains the above-mentioned recombinant type I collagen α1 chain polypeptide encoding gene.

[0014] A recombinant Pichia pastoris engineered bacterium contains the above-mentioned recombinant type I collagen α1 chain polypeptide encoding gene.

[0015] As one of the preferred embodiments of the present invention, the construction method of the engineering bacteria is as follows: the recombinant expression vector constructed by the recombinant type I collagen α1 chain polypeptide encoding gene is linearized and then transferred into Pichia pastoris GS115.

[0016] A method for preparing the above-mentioned recombinant type I collagen α1 chain polypeptide comprises the following steps:

[0017] (1) synthesizing the nucleotide sequence encoding the recombinant type I collagen α1 chain polypeptide shown in SEQ ID NO.1;

[0018] (2) Designing PCR primers to amplify the DNA fragment encoding the recombinant type I collagen α1 chain polypeptide, the primers are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0019] (3) constructing a recombinant expression vector containing the DNA fragment encoding the recombinant type I collagen α1 chain polypeptide;

[0020] (4) After linearizing the constructed recombinant expression vector, transform the competent cells of Pichia pastoris GS115, use geneticin (G418) resistance as a screening marker, screen the transformants, and obtain the recombinant Pichia pastoris engineered bacteria;

[0021] (5) culturing the above-mentioned recombinant Pichia pastoris to express recombinant type I collagen α1 chain polypeptide;

[0022] (6) Isolating and purifying the recombinant type I collagen α1 chain polypeptide expressed in step (5).

[0023] As one of the preferred embodiments of the present invention, in step (3), the expression vector is pPIC9K. Using pPIC9K as the expression vector can continuously secrete and express collagen in the life activities of the engineered bacteria, and the fermentation process is simple and has less impurities, providing an advantageous strain for large-scale fermentation production.

[0024] As one of the preferred embodiments of the present invention, in the step (4), the host is the Pichia pastoris GS115 strain; using Pichia pastoris GS115 as a host to produce recombinant human type I collagen can fully support the biological functions of the collagen products produced using this collagen.

[0025] As one of the preferred embodiments of the present invention, in step (6), ammonium sulfate precipitation, dialysis (dialysis fluid: 10 mM sodium acetate, pH 4.5), ion exchange, and secondary dialysis (dialysis fluid: PBS, pH 7.0) are used to purify the recombinant type I collagen α1 chain polypeptide protein.

[0026] An application of the above recombinant type I collagen α1 chain polypeptide in the field of medical cosmetology and cosmetics.

[0027] The advantages of the present invention compared to the prior art are:

[0028] (1) The recombinant type I collagen α1 chain polypeptide screened and designed by the present invention has a small molecular weight, contains two GER integrin recognition sites (the GER site can be recognized by the integrin recognition site on the cell membrane to promote cell proliferation and adhesion), has excellent activity, has a certain migration effect in human immortalized epidermal cells, and can be widely used in the fields of medical cosmetology, cosmetics, etc.;

[0029] (2) According to the expression characteristics of Pichia pastoris, the present invention performs codon optimization on the nucleotide sequence encoding the recombinant type I collagen α1 chain polypeptide, so that the nucleotide sequence encoding the recombinant type I collagen α1 chain polypeptide can better adapt to the expression system of Pichia pastoris, and finally screens a strain of high-yield recombinant human type I collagen protein through liquid pilot expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a diagram showing the GER active site in the amino acid sequence of the recombinant type I collagen α1 chain polypeptide COLI-9 in Example 1;

[0031] Figure 2 This is the agarose gel electrophoresis diagram of the DNA fragment of the recombinant type I collagen α1 chain polypeptide COLI-9 in Example 1 (in the figure, "M" is a marker, lane "1" is a sample; the target band size is 261 bp);

[0032] Figure 3 This is the electrophoresis diagram of the recombinant type I collagen α1 chain polypeptide COLI-9 protein in Example 4 (in the figure, "M" is a molecular weight marker, lane "1" is the protein in the fermentation broth supernatant, the protein expression level is 280 mg / L, lane "2" is the protein obtained after precipitation by 80% saturated ammonium sulfate, lane "3" is the protein obtained after ion exchange chromatography, and lane 4 is BSA; the molecular weight of the COLI-9 protein detected by electrophoresis is about 9 kDa);

[0033] Figure 4 This is a graph showing the effect of the recombinant type I collagen α1 chain polypeptide COLI-9 on the migration rate of human immortalized epidermal cells in Example 5 (in the graph, Figure A is a microscopic photograph of a scratch test; Figure B is the 24h migration result of human immortalized epidermal cells; and wherein, “**” indicates P<0.01). DETAILED DESCRIPTION

[0034] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0035] The Pichia pastoris GS115 strain and expression vector pPIC9K used in the following examples are both strains and vectors that can be directly purchased. The medium formula used is as follows:

[0036] 1) YPD complete medium:

[0037] Prepare 1L, add 10g yeast extract and 20g peptone to 900mL water, sterilize by high pressure, and then add 100mL sterile 10X glucose solution. The solid medium contains 1.5% agar.

[0038] 2) MD medium:

[0039] To prepare 1L, first autoclave 800mL of water, cool to 60°C, add 100mL of sterile 10XYNB, 2mL of 500X biotin, and 100mL of 10X glucose. The solid medium contains 2% agar.

[0040] 3) BMGY medium:

[0041] Prepare 1L, add 10g of yeast extract and 20g of peptone to 700mL of water, sterilize by high pressure, cool to room temperature, and then add sterile 100mL of 1M potassium phosphate buffer (pH 6.0), 100mL of 10XYNB, 2mL of 500X biotin, and 100mL of 10X glycerol.

[0042] 4) BMMY medium:

[0043] Prepare 1L, add 10g of yeast extract and 20g of peptone to 700mL of water, sterilize by high pressure, cool to room temperature, and then add sterile 100mL of 1M potassium phosphate buffer (pH 6.0), 100mL of 10XYNB, 2mL of 500X biotin, and 100mL of 10X methanol.

[0044] Meanwhile, the DMEM basal culture medium, PBS, fetal bovine serum (FBS), and trypsin used in the following examples are from Vivacell, and the calf serum (BCS), and human immortalized epidermal cell line HaCaT are from Oricell.

[0045] Example 1

[0046] Recombinant type I collagen α1 chain polypeptide COLI-9 encoding gene:

[0047] The present invention finally screened and optimized the coding gene of the recombinant type I collagen α1 chain polypeptide COLI-9, whose nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2 (the GER active site in the amino acid sequence of the recombinant type I collagen α1 chain polypeptide COLI-9 is shown in Figure 1 ).

[0048] Gene screening optimization process:

[0049] (1) Based on the human type I collagen gene sequence in GeneBank, an original sequence fragment containing two "GER integrin recognition site" sequences was found, as shown in SEQ ID NO.5.

[0050] (2) Based on the codon preference of Pichia pastoris and the AT content of the DNA sequence, online codon optimization software was used to optimize the corresponding type I collagen codons to the codons preferred by Pichia pastoris without changing the protein coding sequence. The optimized gene sequence is shown in SEQ ID NO.1. The optimized gene sequence was commissioned to Shanghai Shenggong Biotechnology Co., Ltd. for gene synthesis.

[0051] Gene cloning:

[0052] Specific primers were designed, as shown in SEQ ID NO.3 and SEQ ID NO.4; PCR was used to amplify a DNA fragment encoding the recombinant type I collagen α1 chain polypeptide COLI-9.

[0053] 1% agarose gel electrophoresis was used to detect the target fragment. The results were as follows Figure 2 As shown (target band size is 261 bp).

[0054] Example 2

[0055] Construction of recombinant expression vector pPIC9K-COLI-9:

[0056] An EcoRI restriction site sequence was added to the 5' end of the coding gene sequence, and a stop codon and a NotI restriction site sequence were added to the 3' end; subsequently, the sequence fragment was inserted between the EcoRI and NotI restriction sites of the pPIC9K expression vector to construct an expression vector capable of expressing recombinant type I collagen α1 chain polypeptide.

[0057] Example 3

[0058] Construction of recombinant Pichia pastoris engineering bacteria:

[0059] (1) The successfully constructed recombinant expression vector plasmid pPIC9K-COLI-9 was linearized and transformed into Pichia pastoris GS115 competent cells, spread on MD plates, and cultured in a constant temperature incubator at 30°C for 72 h.

[0060] (2) Pick a single colony from the MD plate and culture it in YPD medium containing 0.75 mg / mL G418 at 30°C and 240 rpm for 18 to 24 h; then transfer it to YPD liquid medium with increasing G418 concentrations for culture.

[0061] (3) Select a strain with good growth potential for fermentation, the steps are as follows:

[0062] Inoculate the bacterial solution into 10mL / 50mL BMGY at a 2% inoculum volume and culture at 30°C, 240rpm, with shaking for 18-24h;

[0063] After centrifugation, the supernatant was discarded, and 10 mL of BMMY was added for resuspending. The cells were cultured with shaking at 28°C and 240 rpm for 48 h, and 100% methanol was added every 24 h to a final concentration of 1.5%;

[0064] The mixture was centrifuged and the supernatant was analyzed by non-reducing SDS-PAGE to obtain the recombinant Pichia pastoris with high expression level.

[0065] Example 4

[0066] Production, preparation and purification of recombinant type I collagen α1 chain polypeptide COLI-9:

[0067] (1) The successfully constructed recombinant Pichia pastoris (glycerol strain) was activated in YPD (G418 containing corresponding resistance) medium and cultured at 30°C and 240 rpm with shaking for 18 h.

[0068] (2) 2% inoculum was transferred to 200 mL / 1 L of BMGY and cultured at 30°C, 240 rpm, with shaking for 16 h until OD 600 Equals 8.

[0069] (3) Centrifuge and discard the supernatant; resuspend the cells in 200 mL of BMMY and culture at 30°C, 240 rpm for 48 h; add anhydrous methanol every 24 h to a final concentration of 1.5%.

[0070] (4) After fermentation, the supernatant was filtered through a 0.45 μm water filter membrane, 80% saturated ammonium sulfate was added, stirred at 0°C for 20 min, allowed to stand for 2 h, and then centrifuged at 12000 rpm for 20 min to collect the precipitate; the precipitate was reconstituted with 10 mM sodium acetate, and the recombinant type I collagen α1 chain polypeptide COLI-9 was isolated and purified by dialysis (dialysis fluid: 10 mM sodium acetate, pH 4.5), ion exchange chromatography, and secondary dialysis (dialysis fluid: PBS, pH 7.0).

[0071] Figure 3 The electrophoresis diagram of the protein obtained by separation and purification of the recombinant type I collagen α1 chain polypeptide COLI-9. The purity of the COLI-9 protein solution after separation and purification was detected by non-reducing SDS-PAGE, and the purity was >95%.

[0072] Example 5

[0073] Bioactivity detection of recombinant type I collagen α1 chain polypeptide COLI-9:

[0074] Scratch test to measure the effect of recombinant type I collagen α1 chain polypeptide COLI-9 on promoting migration of “human immortalized epidermal cells”:

[0075] (1) Cell culture: Use a marker pen to draw 5 horizontal lines on the back of each well of a 6-well plate, with each line spaced approximately 0.5 cm apart as markers; select HaCaT cells, set up a blank group, a control group, and an experimental group, and after digestion, centrifugation, and resuspension, dilute to 2 × 10 6 Each well was 2 mL of cell suspension to ensure that the cell density of each group was the same and that the confluence reached 95-100% after 24 h of culture.

[0076] (2) Scratch test: After 24 h of cell culture, a 200 μL pipette tip was used to scratch the cell layer vertically close to the plane of the culture plate to form a scratch; the cells were rinsed with PBS three times, the scratched cells were removed, and 2 mL of culture medium containing COLI-9 protein was added to 6 wells respectively, with final concentrations of 0.5 mg / mL, 0.05 mg / mL, 0.005 mg / mL, and 0.0005 mg / mL; only culture medium was added to the blank group; commercially available recombinant collagen (Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., [Batch No.] DA2390S007) and fish skin collagen were added to the control group.

[0077] (3) Culture the cells in an incubator at 37°C, 5% CO2, and 95% humidity, and take microscopic photos at a fixed position after 0 h and 24 h.

[0078] (4) Use Image J to measure the scratch area: Use the software to process the scratch images (0h, 24h) and finally obtain the cell migration rate.

[0079] Test results such as Figure 4 As shown. Figure 4 It can be seen that the 24h migration rate of the blank control group (NC) was 21%; the 24h migration rate of the experimental group added with 0.0005 mg / mL COLI-9 was 34%.

[0080] In summary, the recombinant type I collagen α1 chain polypeptide COLI-9 of the present invention has excellent activity, exhibits excellent promotion of cell migration in human immortalized epidermal cells, and can be widely used in the fields of medical cosmetology, cosmetics, etc.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant type I collagen α1 chain polypeptide, characterized in that: The amino acid sequence of the recombinant type I collagen α1 chain polypeptide is shown in SEQ ID NO.

2.

2. The recombinant type I collagen α1 chain polypeptide according to claim 1, characterized in that: The recombinant type I collagen α1 chain polypeptide contains two "GER integrin recognition sites".

3. A recombinant type I collagen α1 chain polypeptide encoding gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO. 1, which is used to encode the recombinant type I collagen α1 chain polypeptide according to claim 1.

4. The recombinant type I collagen α1 chain polypeptide encoding gene according to claim 3, characterized in that: The nucleotide sequence shown in SEQ ID NO.1 is a sequence after codon optimization according to the codon preference of Pichia pastoris.

5. A recombinant expression vector, characterized in that: Contains the recombinant type I collagen α1 chain polypeptide encoding gene according to claim 3 or 4.

6. A recombinant Pichia pastoris engineered bacterium, characterized in that: Contains the recombinant type I collagen α1 chain polypeptide encoding gene according to claim 3 or 4.

7. The recombinant Pichia pastoris engineered bacterium according to claim 6, characterized in that: The construction method of the engineering bacteria is as follows: the recombinant expression vector constructed by the recombinant type I collagen α1 chain polypeptide encoding gene is linearized and then transferred into Pichia pastoris. GS115 .

8. A method for preparing the recombinant type I collagen α1 chain polypeptide as claimed in claim 1, characterized in that: The steps include: (1) synthesizing the nucleotide sequence encoding the recombinant type I collagen α1 chain polypeptide shown in SEQ ID NO.1; (2) Designing PCR primers to amplify the DNA fragment encoding the recombinant type I collagen α1 chain polypeptide, the primers are shown in SEQ ID NO.3 and SEQ ID NO.4; (3) constructing a recombinant expression vector containing the DNA fragment encoding the recombinant type I collagen α1 chain polypeptide; (4) After linearization, the constructed expression vector is transferred into Pichia pastoris to obtain recombinant Pichia pastoris engineered bacteria; (5) culturing the above-mentioned recombinant Pichia pastoris to express recombinant type I collagen α1 chain polypeptide; (6) Isolating and purifying the recombinant type I collagen α1 chain polypeptide expressed in step (5).

9. The method for preparing the recombinant type I collagen α1 chain polypeptide according to claim 8, characterized in that: In the step (6), ammonium sulfate precipitation, dialysis, ion exchange, and secondary dialysis are used to separate and purify the recombinant type I collagen α1 chain polypeptide protein.

10. Use of the recombinant type I collagen α1 chain polypeptide as claimed in claim 1 or 2 in the preparation of cosmetics.

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