Triple-helix recombinant collagen as well as preparation method and application thereof

Abstract

The invention discloses triple helix recombinant collagen as well as a preparation method and application thereof, and belongs to the technical field of gene engineering and protein engineering. According to the invention, on the basis of the amino acid sequence of natural collagen, the amino acid sequence is designed by means of bioinformatics and the like, a novel triple-helix recombinant collagen is constructed, and the triple-helix recombinant collagen has a good triple-helix structure, biocompatibility and biological activity, and can be used for preparing the collagen. The proliferation, the adhesion and the migration of the L929 cells can be obviously promoted, and the method has a wide application prospect in various fields such as food, medicines, cosmetics or instrument products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and protein engineering, and specifically relates to a triple-helix recombinant collagen protein, a coding gene thereof, and a preparation method and application thereof. BACKGROUND

[0002] Collagen is a family of proteins with a unique triple-helix structure in the extracellular matrix, and is the most abundant structural and functional protein in the human body. As a new type of biomaterial, recombinant collagen is increasingly concerned in the fields of dermatology and bone repair. Collagen self-assembles into a fibrous network structure in vivo, providing a biological scaffold for tissues and organs, and participating in the regulation of basic physiological activities such as cell proliferation, migration, and differentiation. Collagen is widely used in tissue engineering and regenerative medicine due to its excellent biocompatibility, biodegradability, and bioabsorbability. Although natural collagen has many important functions, there are some limitations in directly extracting natural collagen from animal tissues. First, animal-derived collagen has immunogenicity problems because the collagen of different species differs in amino acid sequence and structure, and the human immune system will produce an immune response, thereby limiting its application in the medical field. Second, the extraction process of natural collagen is complex, and the yield is limited, making it difficult to meet the demand of large-scale production and application. In addition, the physical and chemical properties of natural collagen are relatively fixed, and it is difficult to customize and optimize according to specific needs.

[0003] In order to overcome the limitations of natural collagen, recombinant collagen technology has emerged. Recombinant collagen is obtained by introducing the gene encoding collagen into a suitable host cell for expression, thereby obtaining collagen with a specific amino acid sequence and structure. Compared with natural collagen, recombinant collagen has many advantages, such as low immunogenicity, strong customizability, and controllable production process. Although there have been some researches and reports on recombinant collagen, the existing recombinant collagen still has some shortcomings in structure and function. For example, the triple-helix structure of some recombinant collagen is not stable enough, resulting in low biological activity; the expression amount of some recombinant collagen is low, making it difficult to meet the demand of large-scale production; in addition, the existing preparation method of recombinant collagen may have complex process, high cost, and other problems. SUMMARY

[0004] In view of the problems of poor water solubility of collagen, large batch differences, serious disease transmission hazards, and insufficient biological activity and stability of recombinant collagen in the prior art, the present application aims to provide a triple-helix recombinant collagen protein, a preparation method thereof, and an application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a triple helix recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO. 1, which is obtained by amino acid mutation from the natural full-length amino acid sequence of human collagen type II alpha 1 chain.

[0007] The present application provides a coding gene of a triple helix recombinant collagen, which encodes the triple helix recombinant collagen.

[0008] Further, the nucleotide sequence encoding the triple helix recombinant collagen is shown in SEQ ID NO. 2.

[0009] The present application provides a recombinant expression vector containing the coding gene of the triple helix recombinant collagen, which expresses the triple helix recombinant collagen.

[0010] The plasmid vector of the recombinant expression vector is pPICZalphaA.

[0011] The present application provides a recombinant cell containing the recombinant expression vector.

[0012] The present application provides a recombinant strain containing the coding gene of the triple helix recombinant collagen or the recombinant expression vector.

[0013] The present application provides a preparation method of a triple helix recombinant collagen, which comprises: constructing an expression vector, transferring the expression vector into a recipient strain, screening, inducing expression and purifying, and obtaining.

[0014] The recipient strain is Pichia pastoris CBS7435.

[0015] The induction of expression is methanol induction, the induction temperature is 20-30℃, and the induction time is 100-140h.

[0016] Further, the concentration of methanol is 0.4% (m / v).

[0017] Further, the induction temperature is 25℃, and the induction time is 120h.

[0018] The triple helix recombinant collagen, the coding gene of the triple helix recombinant collagen, the recombinant expression vector, the recombinant cell, or the recombinant strain are used in the preparation of food, medicine, cosmetics or instrument products.

[0019] Compared with the prior art, the technical scheme of the present application has the following beneficial technical effects:

[0020] The triple helix recombinant collagen provided by the application obtains the recombinant collagen with stable triple helix structure through specific amino acid mutation, and the recombinant collagen has good triple helix structure, biocompatibility and biological activity, and can significantly promote the proliferation, adhesion and migration of L929 cells.

[0021] The preparation method of the triple helix recombinant collagen provided by the application is based on the amino acid sequence of natural recombinant collagen, and the amino acid sequence is designed by means of bioinformatics, and a high-efficiency Pichia pastoris expression system of the triple helix recombinant collagen (CO2TH) is constructed. Under the premise of maintaining high biological activity, high-purity (96%) and high-quality CO2TH is prepared, and the recombinant collagen has good triple helix structure, biocompatibility and biological activity, and can significantly promote the proliferation, adhesion and migration of L929 cells. The bacterial endotoxin, residual DNA and Pichia pastoris protein residual amount reach the “Recombinant Collagen” medical device industry standard, and the prepared recombinant collagen passes the medical grade triple helix recombinant collagen quality detection standard and basis, biocompatibility and biological activity, and successfully realizes the large-scale preparation of medical grade triple helix recombinant collagen.

[0022] The application provided by the application has good triple helix structure, biocompatibility and biological activity, can significantly promote the proliferation, adhesion and migration of L929 cells, and therefore has wide application prospects in various fields such as food, medicine, cosmetics or instrument products. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is an SDS-PAGE gel electrophoresis diagram of the recombinant collagen expressed by the recombinant engineering bacteria CBS7435 / pPICZalphaA-CO2TH in the examples. DETAILED DESCRIPTION

[0024] The following examples are used to further illustrate the application, but these examples are only for better understanding of the application and do not limit the scope or implementation principle of the application, and the implementation mode of the application is not limited to the following contents. If not specifically indicated, the used test methods are all conventional methods, and the used raw materials are all commercial products.

[0025] The Pichia pastoris CBS7435 used in the application is purchased from the American Type Culture Collection (ATCC) by Ningbo Mingzhou Biotechnology Co., Ltd., and is also named Komagataella phaffii NRRL Y-11430, ATCC Number: 76273.

[0026] Example 1

[0027] (I) Collagen sequence design

[0028] The optimized CO2TH gene sequence is introduced into the polyclonal site with pPICZalphaA (purchased from Invitrogen Corporation) as the skeleton to obtain pPICZalphaA-CO2TH, which is finally transformed into Pichia pastoris CBS7435. The detailed steps are as follows:

[0029] 1. According to the human collagen type II mature peptide sequence (P02461-1) published by the protein resource database UniProt (website https: / / www.uniprot.org / ), a large number of hydroxyl amino acids are introduced to design a collagen sequence, and the optimized collagen amino acid sequence is shown as SEQ ID NO. 1:

[0030]

[0031] 2. The gene sequence was designed reversely by using online design tool Jcat (http: / / www.jcat.de / ), and the optimized collagen gene was synthesized by Shanghai Sangon Biological Co., Ltd. according to the preferred codons for expression in the host Pichia pastoris. The sequence of the optimized gene is shown in SEQ ID NO. 2:

[0032]

[0033] (II) Construction of recombinant collagen expression strain

[0034] 1. Linearization of plasmid

[0035] The pPICZalphaA-CO2TH plasmid was extracted using a plasmid extraction kit (Suzhou Emyer Biological Technology Co., Ltd.), and 5 μL of restriction endonuclease Sac1 was added for linearization.

[0036] 2. Purification of linearized product

[0037] The linearized pPICZalphaA-CO2TH product was purified using a purification kit (Suzhou Emyer Biological Technology Co., Ltd.), and the specific steps were as follows:

[0038] An equal volume of Buffer GRp was added, mixed well by inverting or vortexing, and the mixture was transferred to a DNA adsorption column and centrifuged at 10,000 x g for 30-60 seconds. The filtrate was discarded, and the column was fitted back into the collection tube. 500 L of DNA rinse solution was added to the column. The column was centrifuged at 12,000 x g for 30-60 seconds, and this step was repeated once. The filtrate was discarded, and the column was fitted back into the collection tube. 500 mL of 80% ethanol was added to the column. The column was centrifuged at 12,000 x g for 30-60 seconds. The filtrate was discarded, and the column was fitted back into the collection tube. The column was centrifuged at 12,000 x g for 5 minutes, the cap of the column was opened, and it was air-dried for 2 minutes to completely remove the ethanol. The column was fitted into a 1.5 mL centrifuge tube, and 20 mL of ultrapure water was added to the center of the column membrane to dissolve the DNA. It was left to stand for 2 minutes, and then centrifuged at 12,000 x g for 1 minute.

[0039] 3. Dephosphorylation treatment

[0040] In order to prevent self-circularization of the vector plasmid DNA, the linearized pPICZalphaA-CO2TH product was treated with calf intestinal alkaline phosphatase (CIP), and the specific operation was as follows: (1) reaction system: linearized product 35 μL, 10x CP buffer 4 ul, CIP ul, ddH2O to 45 μL; (2) control the reaction temperature on the PCR instrument (add wax oil to seal), 37℃, 15min; 50℃, 15min; 56℃, 30min (inactivation); (3) add 5 μL of 0.1M NaOH, incubate at 56℃ for 10min, for inactivation of CIP.

[0041] 4. Purification of dephosphorylated product

[0042] The dephosphorylated linearized pPICZalphaA-CO2TH product was purified according to the method in step 2.

[0043] 5. Preparation of Pichia pastoris CBS7435G competence

[0044] (1) Pick a single yeast colony and inoculate it into a 50mL Erlenmeyer flask containing 5mL of LYPD medium. Incubate overnight at 30℃ and 250-300r / min. (2) Take 100-500μL of the culture and inoculate it into a 2L Erlenmeyer flask containing 500mL of fresh medium. Incubate overnight at 25-31℃ and 250-300r / min until OD reaches 0.50. 600 Reaching 1.2 to 1.4; (3) Centrifuging the cell culture at 1500g for 5 minutes at 4℃, and resuspending the bacterial pellet in 500mL of ice-cold sterile water; (4) Centrifuging according to step (3), and resuspending the bacterial pellet in 250mL of ice-cold sterile water; (5) Centrifuging according to step (3), and resuspending the bacterial pellet in 30mL of ice-cold 1.5mol sorbitol solution; (6) Centrifuging according to step (3), and resuspending the bacterial pellet in 1.5mL of ice-cold 0.5mol sorbitol solution. Note: It can be aliquoted into 40μL portions, frozen, and stored at -80℃ for one month.

[0045] 6. Electroconversion and Transformant Identification

[0046] The recovered linearized plasmid was added to 40 μL of Pichia pastoris CBS7335 competent cells prepared in step 5. (1) The cells were transferred to a pre-cooled 0.2 cm electroporation cuvette. The electroporation cuvette was placed on ice for 15 min, electroporated at 1500 V for 0.8 S, and then quickly placed on ice. 1 mL of pre-cooled sorbitol solution was added to mix the cells. The cells were then transferred to a 1.5 mL sterile EP tube and spread on MD solid plates. The cells were incubated at 30 °C for 2-3 days until single colonies appeared. Transformants were selected for colony PCR identification. According to the different target genes, corresponding upstream and downstream verification primers were designed. The primers are shown below:

[0047] Primer F: 5'-TGAAATTATGGGTTTCAGCATT-3 (as shown in SEQ ID NO.3)

[0048] Primer R: 5'-TCAACTTCTGTTGAACCGCATG-3 (as shown in SEQ ID NO.4)

[0049] (2) Add 50 LTE (Tris-EDTA buffer) to the PCR tube, pick different transformants, put them into the PCR tube, blow them and incubate at 95℃ for 10 min; centrifuge at 2000g for 1 min, and take 5L of supernatant as template for PCR reaction.

[0050] The PCR reaction system (50 μL) is as follows: 25 μL 2XTaq Mix, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 5 μL template, and finally add ddH2O to make up to 50 mL.

[0051] The amplification procedure is: 95°C, 30s denaturation; 62°C, 15s annealing, 72°C extension 1 min, 32 cycles of the whole reaction.

[0052] (3) The PCR product is subjected to nucleic acid electrophoresis, and the band size is analyzed. The actual value is consistent with the theoretical value, and the target strain pPICZalphaA-CO2TH is obtained, that is, the Pichia pastoris strain containing the recombinant collagen protein gene.

[0053] (Three) Preparation of recombinant collagen protein

[0054] 1. Preparation of primary seed

[0055] Take the strain CBS7435 / pPICZalphaA-CO2TH preserved at -80°C for activation, and use a sterilized tooth to dip the bacterial liquid on a YPD solid plate: culture at 30°C for 2-3 days until single colonies appear, pick single colonies in YPD liquid medium, culture at 30°C, 200 rpm for about 24 h, until OD 600 reaches 5-40, which is the primary seed liquid. The seed liquid can be adjusted according to the size of the culture volume.

[0056] 2. Preparation of secondary seed

[0057] Take 300-500 mL of primary seed liquid and inoculate into a 50 L fermenter (the initial fermentation YDD liquid medium is 30 L, culture at 28-40°C, 150-200 r / min under shaking, when the bacterial OD 600 reaches 10.0-12.0, the secondary seed is obtained; automatically add ammonia water or phosphoric acid to control the pH value to 5.2, and adjust the speed, air flow, and tank pressure (0.8 atm) of the fermentation equipment;

[0058] The YDD liquid medium formula is as follows:

[0059] Basic medium: glucose 20 g / L, soybean peptone 20 g / L, yeast powder 10 g / L, potassium hydrogen phosphate 27 mL / L, potassium dihydrogen phosphate 12 mg / L, calcium sulfate 1 g / L, potassium sulfate 18 g / L, sucrose 20 g / L, magnesium sulfate heptahydrate 15 g / L, sodium hydroxide 4 g / L, glycerol 60 g / L (high-temperature sterilization); trace elements: iron chloride 2.0 g / L, potassium iodide 0.08 g / L, manganese sulfate monohydrate 3.0 g / L, zinc chloride 20.0 g / L, ferrous sulfate heptahydrate 65.0 g / L, concentrated sulfuric acid 5 mL / L (filtered and sterilized), and the specified concentration is prepared after filtration and sterilization.

[0060] 3. Fermentation control

[0061] Because the bacteria have been preliminarily adapted to the fermentation medium in the secondary seed culture stage, the bacteria enter the logarithmic phase after a relatively short adaptation period in the fermentation process. At this time, the pH value is automatically controlled, and ammonia water or phosphoric acid is automatically added to control the pH value at 5.2. The rotation speed and the aeration rate are adjusted to maintain the dissolved oxygen (DO) at more than 50y. When the glycerol in the fermentation tank is consumed (about 21 hours after inoculation), the DO value rises sharply, and it is detected that the glycerol has been consumed. At this time, 800 mL of glycerol is added at a rate of 5.5 mL / min, and the feeding is stopped after 7 hours. The DO value rises sharply again after about 2-3 hours, and the amount of the bacteria reaches the predetermined requirement (the wet weight of the bacteria in this experiment is 500-600 g / L).

[0062] 4. Methanol-induced expression

[0063] When the wet weight of the bacteria reaches the predetermined value (250-300 g / L), methanol induction is started, and 80 mL of the first methanol solution (1.2%) is added. Feeding: after the methanol is consumed, 250 mL of methanol is added again at a rate of 2.5 mL / min. When the methanol is consumed, 1.2% methanol aqueous solution is added at a rate of 1.5 mL / min. After 24 hours of induction, 1.2% methanol aqueous solution is added at a rate of 0.5 mL / min. After 12 hours of induction, the fermentation is ended after 36 hours.

[0064] 5. Treatment and purification of the fermentation broth

[0065] After the induction is ended, the fermentation broth is centrifuged at 6000xg for 10 min, and the supernatant is collected. The supernatant containing the recombinant collagen fermentation broth is precipitated by adding 8%-15% saturated ammonium sulfate, and the supernatant is collected by centrifugation at 20000 rpm for 20 min. Then, 2M imidazole solution is added to the supernatant to make the final concentration 20mM, and particles are removed by passing through a 0.45μm filter. Then, the next step of purification is performed by passing through a Ni 2+ affinity chromatography column, and the process is as follows: first, the Ni 2+ affinity chromatography column is equilibrated with pH 7.4 equilibration buffer (50mM phosphate buffer + 0.5M NaCl); then, the filtered supernatant is loaded at a rate of 10 mL / min, and 500 mL of equilibration buffer (50mM phosphate buffer + 0.5M NaCl + 20mM imidazole) is used to wash the unabsorbed sample; then, 100 mL of elution buffer (pH 7.4, 50mM phosphate buffer + 0.5M NaCl + 500mM imidazole) is used to elute the purified sample; finally, the molecular sieve G25 (GE Company) is used to obtain the recombinant collagen stock solution (stock solution (1)) Figure 1 ) dissolved in 50mM pH 5.5 phosphate buffer. Figure 1It can be seen that a clear band appears near 118KD, which indicates that after a series of fermentation broth treatment and purification steps, a relatively pure recombinant collagen protein is successfully obtained.

[0066] Example 2 Quality detection of recombinant collagen protein

[0067] Based on Example 1, the quality of the recombinant collagen protein obtained by the present application was detected according to the quality detection standards and basis of medical grade triple helix recombinant collagen protein (see Table 1).

[0068] Table 1: Quality detection standards and basis of medical grade recombinant collagen protein

[0069]

[0070] Note: According to the "Recombinant Collagen Protein" of the People's Republic of China Pharmaceutical Industry Standard YYT1849-2022.

[0071] ①Purity: The purity of the recombinant collagen protein was characterized by SDS-PAGE. The separation gel was prepared according to the ratio. Inject the gap between the concave glass plates, and seal with water. After the gel is formed, discard the water and inject the concentrated gel. Carefully insert the sample slot into the concentrated gel and let it stand to polymerize the gel. Install the electrophoresis tank, add electrode buffer, add triple helix recombinant collagen protein sample, set the voltage to 60V, and when the sample enters the concentrated gel, adjust the voltage to 120V. When the bromophenol blue migrates to the bottom of the gel, stop electrophoresis. Take out the gel and place it in excess coomassie brilliant blue staining solution, heat in the microwave for about 2 min, then place it on a shaker for 1-2 h for staining. After the stained gel is washed with water to remove excess dye, it is placed in excess decolorizing solution and heated in a microwave oven for about 2 min, then placed on a decolorizing shaker. Scan and analyze using a gel imaging instrument.

[0072] ②Bacterial endotoxin: According to the instructions of the limulus reagent kit, a standard curve is drawn. Prepare positive control of test sample (take a certain volume of 1 EU / mL working standard solution, add an equal volume of test sample solution and mix well, finally prepare a solution containing 0.5 EU / mL working standard), negative control of test sample (bacterial endotoxin test water) and test sample (dilute the test sample with bacterial endotoxin test water by an appropriate multiple). Take the instrument special detection tube, add 0.1 mL of reconstituted limulus reagent for use. Then add 0.1 mL of test sample solution, positive control solution, make two tubes in parallel, and place them in the reactor for detection.

[0073] ③Residual DNA: Reagents were prepared according to the PicoGreen fluorescence kit instructions. The triple-helical recombinant collagen sample was diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 100 μL of the prepared standard working solution and the sample solution to be tested were added to the enzyme-labeled plate, and then 100 μL of Quant-iT TM Pico The working solution of the reagent was added to each well of the enzyme-labeled plate and mixed with the standard and sample to be tested, and was placed in the dark at room temperature for 5 minutes. Measurement was performed at a wavelength of 520 nm.

[0074] ④Pichia pastoris protein residue: 25 μL of the triple-helical recombinant collagen solution was taken and placed in the specified microplate, and 100 μL of HRP-labeled Anti-E. coli was added to each well. The microplate was sealed with a sealing film and placed on a constant temperature shaker at a speed of 600 rpm for 90 min; after the reaction was completed, the reaction was discarded and the residual liquid was removed by tapping on a water-absorbing paper and washing. The above four steps were repeated four times. 100 μL of TMB substrate was added to each well. It was allowed to react at room temperature for 30 min. Finally, 100 μL of stop solution was added. The absorbance was read at a wavelength of 450 nm.

[0075] Example 3

[0076] This example is based on Example 1, and the triple-helical structure of the recombinant collagen obtained by the present application is characterized.

[0077] A recombinant collagen sample with a concentration of 1 mg / mL was prepared using a pH 7.0, 10 mM phosphate buffer (PBS), and the solution was equilibrated at 4°C for at least 24 h. The circular dichroism of the sample was determined using a Chirascan CD equipped with a Peltier temperature controller. The wavelength scan range was 190-260 nm, with an interval of 0.5 nm, and the average time was 5 s. A significant negative peak (negative Cotton effect) appeared near 220 nm, reflecting the n→π* electronic transition of the recombinant protein peptide chain (transition of the lone pair electron of the amide bond to the π* antibonding orbital); a positive peak appeared at 198 nm, reflecting the PPII helix characteristic of a single peptide chain in a triple helix. The 198 nm positive peak and the 220 nm negative peak together constitute the "double peak" pattern of collagen, indicating that the recombinant collagen prepared in Example 1 has a triple-helical structure.

[0078] Example 4

[0079] This example is based on Example 1, and the biocompatibility and biological activity of the recombinant collagen obtained by the present application are detected.

[0080] 3

[0081] 6

[0082] 5

[0083] ​​​​​​(4) Cell migration: the ability of recombinant collagen to promote L929 cell migration was detected by cell scratch method. Three horizontal lines were drawn on the bottom of a 6-well TC-treated plate using a marker pen, with a 0.5-1 cm interval between each line. 6 x 10 5 cells were added to each well, and cultured for 4 hours. A 10 μL gun head was used to draw a line vertically along the horizontal line on the bottom, forming a cell scratch. The cells were washed with PBS for 3 times to remove the detached cells. Using DMEM medium as a control group, 2 mL of recombinant collagen solution was added to the 6-well plate, and cultured at 37°C and 5% CO2. The plate was taken out at 0 h, 24 h and 48 h, respectively, and the scratch area was observed under an inverted microscope. The specific detection results are shown in Table 2.

[0084] Table 2: Detection results of recombinant collagen of the present application

[0085]

[0086] As can be seen from the data in Table 2, through microscopic observation, the cells treated with recombinant collagen were completely spread and had abundant pseudopodia, while the cells treated with natural collagen were unevenly spread and had abundant pseudopodia, which is an important marker for good cell growth and migration, indicating that recombinant collagen can provide a more suitable growth environment for cells, and its biocompatibility is better than that of natural collagen. Using the CCK-8 detection method, the cell activity of the recombinant collagen group was 98 ± 1.2%, and that of the natural collagen group was 84.7 ± 2.8%. The cell activity of the recombinant collagen group was significantly higher, indicating that the recombinant collagen had little toxicity to the cells and almost did not affect the normal growth and metabolism of the cells, and performed better in safety. The CCK-8 (48h) detection results showed that the cell proliferation rate of the recombinant collagen group was 152.4 ± 5.6%, and that of the natural collagen group was 112.5 ± 3.9%. The cell proliferation rate of the recombinant collagen group was significantly higher, indicating that the recombinant collagen could more effectively stimulate cell proliferation and promote the increase in cell number. The crystal violet staining counting method showed that the number of cells adhered to the recombinant collagen group was 286 ± 15, and that of the natural collagen group was 172 ± 10. The number of cells adhered to the recombinant collagen group was significantly higher, indicating that the recombinant collagen could better promote the adhesion of cells to the matrix, which was conducive to the positioning and growth of cells in tissues. The scratch test results showed that the cell migration rate of the recombinant collagen group was 92.5 ± 3.8%, and that of the natural collagen group was 62.1 ± 5.0%. The cell migration rate of the recombinant collagen group was significantly faster, further verifying the microscopic image observation results, indicating that the recombinant collagen had obvious advantages in promoting cell migration, which was crucial for wound healing, tissue repair and the like.

[0087] In summary, the recombinant collagen prepared by the application has significant advantages in biocompatibility, non-cytotoxicity, promotion of cell proliferation, adhesion and migration, etc., fully meets the quality detection standards of the People's Republic of China Pharmaceutical Industry Standard YYT1849-2022 'Recombinant Collagen Protein', has good biocompatibility, non-cytotoxicity, and can significantly promote the proliferation, adhesion and migration of L929 cells.

[0088] The above examples are only exemplary embodiments of the application and are not intended to limit the application. The scope of protection of the application is defined by the claims. The above content is only an example and a description of the concept of the application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace, as long as they do not deviate from the concept of the application or exceed the scope defined by the claims, which should belong to the protection scope of the application.

Claims

1. A triple-helix recombinant collagen, characterized in that, The amino acid sequence of the triple-helix recombinant collagen is shown in SEQ ID NO.1, and it is obtained by amino acid mutation from the natural full-length amino acid sequence of the human type II collagen α1 chain.

2. A gene encoding a triple-helix recombinant collagen, characterized in that, The encoding gene encodes the triple-helix recombinant collagen as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene of the triple-helix recombinant collagen as described in claim 2, and expresses the triple-helix recombinant collagen as described in claim 1.

4. The recombinant expression vector as described in claim 3, characterized in that, The plasmid vector for the recombinant expression vector is pPICZalphaA.

5. A recombinant cell, characterized in that, The recombinant cell described herein contains the recombinant expression vector as described in claim 3 or 4.

6. A recombinant bacterial strain, characterized in that, The recombinant strain contains the encoding gene for the triple-helix recombinant collagen as described in claim 2 or the recombinant expression vector as described in any one of claims 3 to 4.

7. The method for preparing triple-helix recombinant collagen according to claim 1, characterized in that, include: An expression vector was constructed, which was then transformed into a recipient strain. The strain was screened, induced to express, and purified to obtain the final product.

8. The method for preparing triple-helix recombinant collagen according to claim 7, characterized in that, The recipient strain was Pichia pastoris CBS7435.

9. The method for preparing triple-helix recombinant collagen according to claim 7, characterized in that, The induced expression was achieved by methanol induction at a temperature of 20–30°C for 100–140 h.

10. The use of a triple-helix recombinant collagen protein according to claim 1, or the encoding gene of a triple-helix recombinant collagen protein according to claim 2, or a recombinant expression vector according to any one of claims 3 to 4, or a recombinant cell according to claim 5, or a recombinant strain according to claim 6 in the preparation of food, pharmaceutical, cosmetic or medical device products.

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