Recombinant human type II collagen fragment and gene, vector, bacteria and application

Abstract

The present invention provides a recombinant human type II collagen fragment, the amino acid sequence is shown in SEQ ID NO.2, and the coding gene sequence is shown in SEQ ID NO.1. The present invention also provides an expression vector and Pichia pastoris engineering bacteria comprising the above-mentioned recombinant human type II collagen fragment coding gene. At the same time, the present invention also provides a preparation and application of the above-mentioned recombinant human type II collagen fragment. The recombinant human type II collagen fragment of the present invention not only has a significant effect on promoting the proliferation and migration of human chondrocytes, but also shows excellent effects of promoting cell migration and promoting adhesion in human immortalized epidermal cells and mouse embryonic fibroblasts, and can be widely used in artificial bones, artificial cartilage, artificial corneas, vitreous bodies, wound repair materials, supporting biomaterials, medical cosmetic surgery, cosmetics and other fields.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant human type II collagen fragment and gene, an expression vector, bacteria and applications. Background Art

[0002] Collagen is an extremely important structural protein in the human body, with special biological functions, rich content and a wide variety of types, designed to support organs and protect tissues. Human collagen has weak antigenicity, has the function of inducing cell proliferation, differentiation and promoting cell adhesion, can maintain cell structure, provide vascular scaffolds, etc. Its degradation product, collagen hydrolyzate, has good antioxidant function, so it has become an important material in the fields of wound dressing, cosmetics, tissue engineering, and medical devices.

[0003] There are as many as 29 types of collagen, and the most widely studied are type I, II and III collagen. Among them, type II collagen is widely distributed in connective tissues such as cartilage, vitreous and skin. It has strong tensile and compressive resistance and has been widely used as a biomaterial in fields such as tissue engineering and regenerative medicine. This type of collagen has a total length of 1014 amino acids and a theoretical molecular weight of 91.4kDa. It is composed of 3 α1 chains, each of which forms a right-handed helical structure, and the 3 α1 chains are further entangled to form a left-handed helix. In articular cartilage, type II collagen can maintain the elasticity and toughness of articular cartilage; in the eyeball, type II collagen can maintain the structure of the eyeball and the transparency of the vitreous.

[0004] At present, the production of type II collagen is mainly obtained through animal tissue extraction. For example, type II collagen is extracted from the cartilage of pigs, cattle, sheep and other animals. However, collagen from animal sources is highly antigenic, the body's rejection reaction is severe, and there are safety risks such as viruses and infectious diseases.

[0005] With the continuous advancement of genetic engineering technology, the technology for producing recombinant humanized collagen has gradually matured, including expression systems with animals, insect cells, Escherichia coli and Pichia pastoris as hosts. For example, Sun Jingchen et al. constructed a baculovirus multi-gene expression system containing a recombinant plasmid, used Escherichia coli to infect insect cells to produce virus fluid, and transfected the virus fluid into insect cells or directly injected into silkworm larvae to produce recombinant human type II collagen. Nokelainen M et al. co-infected insect cells with two baculoviruses to produce cartilage-specific type II collagen with a stable triple helix. However, the animal and insect cell expression systems have high production costs, long cycles, and low yields, which are limited to laboratory levels and cannot be mass-produced. At the same time, some researchers use Escherichia coli as an expression host to recombinantly express human type II collagen peptides, and use Escherichia coli to express human type II collagen functional polypeptides. However, the protein expression amount of the Escherichia coli expression system is limited, lacks post-translational modification, has endotoxins, and the purification steps are cumbersome, increasing production costs.

[0006] Using Pichia pastoris as a host to express recombinant humanized collagen has the advantages of high yield, no endotoxin, short production cycle, high-density fermentation, and low cost. Qian Song et al. successfully used Pichia pastoris to secrete and express the full-length peptide chain of human type II collagen, and added specific affinity purification tags at the N-terminus and C-terminus respectively. The fermentation supernatant was purified by nickel column to obtain the full-length α1 chain of type II collagen with biological activity. However, the molecular weight of full-length collagen is too large, the transdermal effect is poor, and the onset time and onset concentration are limited by practical applications. In comparison, collagen fragments can penetrate the stratum corneum and dermis more effectively, have strong free radical scavenging activity, lipid peroxidation inhibition ability, oxidation and oxidative damage DNA protection activity, can downregulate intracellular ROS accumulation, and play a role in extending the life of skin cells, inhibiting peroxidation, inhibiting the activity of tyrosine, improving skin immunity, improving skin water retention, promoting collagen synthesis and dermal cell hyaluronic acid synthesis, etc. However, at present, there are few studies on highly active collagen fragments of human type II collagen. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant human type II collagen fragment and gene, an expression vector, bacteria and application.

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

[0009] A recombinant human type II collagen fragment, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the recombinant human type II collagen fragment contains one "tripeptide signal recognition site RGD", two "GER" and three "GEK integrin recognition sites".

[0011] A gene encoding a recombinant human type II collagen fragment, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and is used to encode the recombinant human type II collagen fragment according to claim 1 or 2.

[0012] As one of the preferred embodiments of the present invention, the nucleotide sequence shown in SEQ ID NO.1 is a codon-optimized sequence adapted to the Pichia pastoris expression system.

[0013] An expression vector comprises the coding gene of the above-mentioned recombinant human type II collagen fragment.

[0014] As one of the preferred embodiments of the present invention, the method for constructing the expression vector is as follows: adding an EcoRI restriction site sequence to the 5' end of the coding gene sequence, and adding a stop codon and a NotI restriction site sequence to the 3' end; then, inserting the sequence fragment into the pPIC9K expression vector through the EcoRI and NotI restriction sites to obtain the corresponding recombinant expression vector.

[0015] A recombinant Pichia pastoris engineered bacterium comprises the coding gene of the above-mentioned recombinant human type II collagen fragment.

[0016] As one of the preferred embodiments of the present invention, the construction method of the recombinant Pichia pastoris engineered bacteria is as follows: the expression vector constructed by the coding gene of the recombinant human type II collagen fragment is linearized and then transferred into Pichia pastoris GS115.

[0017] A method for preparing the above-mentioned recombinant human type II collagen fragment comprises the following steps:

[0018] (1) synthesizing the nucleotide sequence encoding a recombinant human type II collagen single chain as shown in SEQ ID NO.1;

[0019] (2) Designing PCR primers to amplify and obtain a DNA fragment encoding a single-chain recombinant human type II collagen;

[0020] (3) constructing a recombinant expression vector comprising the DNA fragment encoding the recombinant human type II collagen single chain, wherein the expression vector is pPIC9K;

[0021] (4) After linearization, the constructed expression vector was transformed into Pichia pastoris GS115 to obtain recombinant Pichia pastoris engineered bacteria;

[0022] (5) culturing the above-mentioned recombinant Pichia pastoris to express recombinant human type II collagen fragment;

[0023] (6) Isolation and purification of the recombinant human type II collagen fragment expressed in step (5): dialysis, ion exchange, and secondary dialysis are used to separate and purify the recombinant type II collagen fragment.

[0024] An application of the above-mentioned recombinant human type II collagen fragment in the fields of artificial bone, artificial cartilage, artificial cornea, vitreous body, wound repair materials, supporting biomaterials, medical cosmetic surgery, cosmetics, etc.

[0025] The advantages of the present invention compared to the prior art are:

[0026] (1) The recombinant human type II collagen fragment provided by the present invention contains one "tripeptide signal recognition site RGD", two "GER" and three "GEK integrin recognition sites" (GER and GEK sites can be recognized by integrin recognition sites on the cell membrane, and then transmitted to cells through signal transduction to promote cell proliferation and adhesion; RGD site can be recognized by integrin recognition sites on the cell membrane, and then transmitted to cells through signal transduction to promote cell adhesion. It is also a tripeptide recognition site in collagen, which helps collagen to form a triple helix structure). It not only has a significant effect on promoting the proliferation and migration of human chondrocytes, but also shows excellent effects of promoting cell migration and promoting adhesion in human immortalized epidermal cells HaCaT and mouse embryonic fibroblasts NIH / 3T3, respectively, and shows good biocompatibility and biological activity. It can be used not only in common fields of type II collagen such as artificial bone materials, cornea, vitreous body, but also in the field of skin repair;

[0027] (2) The present invention optimizes the nucleotide sequence encoding recombinant human type II collagen according to the expression characteristics of Pichia pastoris, making the nucleotide sequence encoding recombinant human type II collagen more suitable for the expression system of Pichia pastoris, and obtains a strain of high-yield recombinant type II collagen through rapid screening by small-scale expression test;

[0028] (3) The present invention purifies recombinant human type II collagen by combining “dialysis and ion exchange chromatography” to obtain recombinant human type II collagen with high recovery rate and high purity;

[0029] (4) The present invention uses Pichia pastoris as an expression host to produce recombinant human type II collagen, which has the advantages of being endotoxin-free, capable of post-translational modification of collagen, and capable of high-density fermentation, and can fully support the biological functions of the collagen products produced using this collagen;

[0030] (5) The expression vector pPIC9K used in the present invention is a secretory expression vector, which can continuously secrete and express collagen in the life activities of the engineered bacteria. The fermentation process is simple and contains less impurities, thus providing an advantageous strain for fermentation production in a fermenter. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the active site map in the amino acid sequence of the recombinant human type II collagen fragment COLII-23 in Example 1 (in the map, RGD is the tripeptide signal recognition site, and GER and GEK are integrin recognition sites);

[0032] Figure 2 This is the protein electrophoresis diagram obtained by separation and purification of the recombinant human type II collagen fragment COLII-23 in Example 4 (in the figure, the first lane is the molecular weight marker, and the second lane is the purified COLII-23 protein; the molecular weight of the COLII-23 protein detected by electrophoresis is about 40 kDa);

[0033] Figure 3 This is a graph showing the effect of recombinant human type II collagen COLII-23 on the proliferation of human chondrocytes in Example 5 (in the graph, "**" indicates P<0.01);

[0034] Figure 4 This is a graph showing the effect of recombinant human type II collagen COLII-23 on the migration rate of human chondrocytes in Example 5 (in the graph, graph A shows the migration rate results at 24 h, and graph B shows the migration rate results at 48 h, and “*” indicates P<0.05);

[0035] Figure 5 This is a microscopic photograph of the scratch test of the recombinant human type II collagen COLII-23 on the migration rate of human immortalized epidermal cells in Example 6;

[0036] Figure 6 This is a graph showing the effect of recombinant human type II collagen COLII-23 on the migration rate of human immortalized epidermal cells in Example 6 (in the graph, Graph A shows the migration rate results at 24 h, Graph B shows the migration rate results at 48 h, "*" indicates P<0.05, and "**" indicates P<0.01);

[0037] Figure 7 This is a graph showing the effect of recombinant human type II collagen COLII-23 on the adhesion of mouse embryonic fibroblasts in Example 7 (in the graph, "*" indicates P<0.05);

[0038] Figure 8 This is a curve chart of the growth of recombinant human type II collagen COLII-23 in a 5L fermenter during high-density fermentation, changes in wet weight of the bacteria, and expression of the target protein in Example 8;

[0039] Fig. 9 This is a scanning electron microscope microstructure observation result of the recombinant human type II collagen COLII-23 in Example 9 (in the figure, Figure A is the observation result at 300 magnifications, and Figure B is the observation result at 500 magnifications). DETAILED DESCRIPTION

[0040] 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.

[0041] The Pichia pastoris GS115 strain used in the following examples was from Shanghai Angyu Company, and the expression vector pPIC9K was from Shanghai Shenggong Company, both of which are directly commercially available strains and vectors. The medium formula used is as follows:

[0042] 1) YPD complete medium (1 L): Add 10 g / L yeast extract and 20 g / L peptone to 900 mL water, sterilize by high pressure, and then add 100 mL sterile 10× glucose solution. The solid medium contains 1.5% agar.

[0043] 2) MD medium (1 L): First, sterilize 800 mL of water by high pressure, cool to 60°C, then add 100 mL of 10XYNB, 2 mL of 500X biotin, and 100 mL of 10X glucose. The solid medium contains 2% agar.

[0044] 3) BMGY medium (1 L): add 10 g / L yeast extract and 20 g / L peptone to 700 mL water, sterilize by autoclaving, cool to room temperature, and then add 100 mL 1 M potassium phosphate buffer (pH 6.0), 100 mL 10XYNB, 2 mL 500X biotin, and 100 mL 10X glycerol.

[0045] 4) BMMY medium (1 L): Add 10 g / L yeast extract and 20 g / L peptone to 700 mL of water, sterilize by autoclave, cool to room temperature, and then add 100 mL 1 M potassium phosphate buffer (pH 6.0), 100 mL 10XYNB, 2 mL 500X biotin, and 100 mL 10X methanol.

[0046] 5) BSM medium: CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, defoaming agent 1 g / L; after sterilization, add 26.7 mL of 85% phosphoric acid.

[0047] 6) PTM1 trace elements: CuSO4·5H2O 6g / L, NaI 0.08g / L, MnSO4·H2O 3g / L, NaMoO4·2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20g / L, FeSO4·7H2O65g / L, biotin 0.2g / L, H2SO4 5mL.

[0048] 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), mouse embryonic fibroblast cell line NIH / 3T3, and human immortalized epidermal cell line HaCaT are from Oricell.

[0049] Example 1

[0050] Recombinant human type II collagen fragment COLII-23 encoding gene:

[0051] The present invention finally screened and optimized the recombinant human type II collagen fragment COLII-23 encoding gene, whose nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2 (the RGD, GER and GEK active sites in the amino acid sequence of the recombinant human type II collagen fragment COLII-23 are shown in Figure 1 ).

[0052] Gene screening optimization process:

[0053] (1) Based on the Genebank gene sequence NM_001844.4 of human type II collagen, an original sequence fragment containing one "tripeptide signal recognition site RGD", two "GER" and three "GEK integrin recognition sites" was found, as shown in SEQ ID NO.3.

[0054] (2) Sequence optimization was performed based on the codon preference of Pichia pastoris and the GC content of the DNA sequence. The optimized gene sequence is shown in SEQ ID NO. 1. The optimized gene sequence was artificially synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0055] Gene cloning:

[0056] Specific primers were designed and PCR was used to amplify the DNA fragment encoding the single-stranded recombinant human type II collagen COLII-23. The PCR system is shown in Table 1.

[0057] Table 1 PCR amplification reaction system and reaction conditions

[0058]

[0059] Detect the target fragment by 1% agarose gel electrophoresis.

[0060] Example 2

[0061] Construction of recombinant expression vector pPIC9K-COLII-23:

[0062] An EcoRI restriction site sequence was added to the 5' end of the coding gene sequence, and a stop codon and NotI restriction site sequence were added to the 3' end; subsequently, the sequence fragment was inserted into the pPIC9K expression vector through the EcoRI and NotI restriction sites to obtain the corresponding recombinant expression vector pPIC9K-COLII-23.

[0063] Example 3

[0064] Construction of recombinant Pichia pastoris engineering bacteria:

[0065] (1) The recombinant expression vector pPIC9K-COLII-23 was linearized and then transferred into the competent cell of Pichia pastoris GS115.

[0066] (2) Single clones were picked from the transformed MD plate and placed in a 48-well culture plate containing 500 μL YPD (0.75 mg / mL G418) medium. The plates were cultured at 30°C and 240 rpm for 18–24 h. The plates were then transferred to YPD liquid medium with increasing G418 content.

[0067] (3) Fermenting the strain with good growth, the steps are as follows:

[0068] 2% inoculum: inoculate the bacterial solution into 10mL / 50mL BMGY and culture at 30℃, 240rpm for 18-24h;

[0069] After centrifugation, the supernatant was discarded and 10 mL of BMMY was added for resuspending. The cells were cultured at 28°C, 240 rpm for 48 h, and 100% methanol was added every 24 h to a final concentration of 1.5%;

[0070] The mixture was centrifuged and the supernatant was taken for SDS-PAGE analysis to obtain the recombinant Pichia pastoris with high expression level.

[0071] Example 4

[0072] Production, preparation and purification of recombinant human type II collagen fragment COLII-23:

[0073] (1) The successfully constructed recombinant Pichia pastoris (glycerol strain) was activated in YPD (G418 containing corresponding resistance) medium at 30°C, 240 rpm, for 18 h;

[0074] (2) 2% inoculum was transferred to 200 mL / 1 L of BMGY and cultured at 30°C, 240 rpm for 26-28 h to completely consume the glycerol in the culture medium;

[0075] (3) Take 200 mL of BMMY to resuspend the cells and culture at 25°C, 240 rpm for 48 h; add 100% methanol every 24 h to a final concentration of 1.5%.

[0076] (4) After fermentation, the supernatant was filtered through a 0.22 μm water filter membrane to obtain a clarified fermentation supernatant, which was then separated and purified by dialysis (dialysis fluid: 20 mM Tris-HCl, pH 8), ion exchange chromatography, and secondary dialysis (dialysis fluid: PBS, pH 7.0).

[0077] Figure 2 This is the protein electrophoresis diagram of the recombinant human type II collagen fragment COLII-23 separated and purified. The COLII-23 protein solution after separation and purification was analyzed by gel filtration chromatography and non-reducing-SDS-PAGE, and the purity was >95% and the recovery rate was >85%.

[0078] Example 5

[0079] Recombinant human type II collagen fragment COLII-23 promotes growth and migration of human chondrocytes C28I2:

[0080] 1. MTT method to measure the effect of recombinant human type II collagen on the proliferation of human chondrocytes:

[0081] (1) Human chondrocytes C28I2 were digested, centrifuged, resuspended, and diluted to 3×10 4 / mL, 100 μL was inoculated into each well of a 96-well cell culture plate; at the same time, culture medium without cells was inoculated as a blank control, and cultured at 37°C, 5% CO2, 95% humidity for 24 h.

[0082] (2) Prepare COLII-23 protein suspensions at 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL, and 0.078125 mg / mL.

[0083] (3) The culture medium was discarded, and the blank control was added with culture medium, the negative control was added with culture medium, the positive control was added with culture medium containing 5% DMSO, and the experimental group was added with culture medium containing different concentrations of protein solution. The cells were cultured at 37°C, 5% CO2, and 95% humidity for 48 h.

[0084] (4) Add 20 μL MTT to each well and incubate in an incubator for 4 h at 37°C, 5% CO2, and 95% humidity. Add 150 μL DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Detect the absorbance at 490°C using an ELISA reader.

[0085] Test results such as Figure 3 As shown. Figure 3 It can be seen that the survival rate of the negative control group was 100%, and the survival rate of the C28I2 experimental group supplemented with COLII-23 was 115%.

[0086] 2. Scratch test to measure the effect of recombinant human type II collagen on promoting the migration of human chondrocytes:

[0087] (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 C28I2 cells and set up a blank group, a control group, and an experimental group. 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.

[0088] (2) Scratch test: After 24 hours of cell culture, a 200 μL pipette tip was used to scratch the cell layer vertically close to the culture plate plane to form a scratch, and the cells were rinsed with PBS 3 times to remove the scratched cells. 2 mL of culture medium containing recombinant human type II collagen COLII-23 was added to 6 wells respectively, with a final concentration of 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.01 mg / mL. The blank group only added culture medium, the control group added fish skin collagen, and the positive control group added 4% FBS (adding serum FBS will definitely promote cell growth).

[0089] (3) The cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity, and microscopic photography was performed at a fixed position after 0 h, 24 h, and 48 h.

[0090] (4) Use Image J to measure the scratch area: Use the software to process the scratch images (0h, 24h / 48h) and finally obtain the cell migration rate.

[0091] 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) is 18%, and the 48h migration rate is 40%; the 24h migration rate of fish skin collagen is 20%, and the 48h migration rate is 36%; the 24h migration rate of the experimental group added with 0.05mg / mL COLI-23 is 33%, and the 48h migration rate is 54%.

[0092] Example 6

[0093] The effect of recombinant human type II collagen COLII-23 on the migration of human immortalized epidermal cells HaCaT (verified by scratch test):

[0094] (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. Use HaCaT cells to set up a blank group, a control group, and an experimental group. 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.

[0095] (2) Scratch test: After 24 hours of cell culture, a 200 μL pipette tip was used to scratch the cell layer vertically close to the culture plate surface to form a scratch. The cells were rinsed with PBS three times, the scratched cells were removed, and 2 mL of culture medium containing recombinant human type II collagen COLII-23 was added to 6 wells respectively, with a final concentration of 0.05 mg / mL, 0.005 mg / mL, and 0.0005 mg / mL. The blank group was only added with culture medium. The control group was added with commercially available recombinant collagen.

[0096] (3) Cultivate in an incubator at 37°C, 5% CO2, and 95% humidity, and take microscopic photos at a fixed position after 0 h, 24 h, and 48 h. Figure 5 shown.

[0097] (4) Use Image J to measure the scratch area: Use the software to process the scratch images (0h, 24h / 48h) and finally obtain the cell migration rate.

[0098] Test results such as Figure 6 As shown. Figure 6 It can be seen that the 24h migration rate of the blank control group (NC) was 23%, and the 48h migration rate was 40%; the 24h migration rate of commercially available recombinant collagen was 25%, and the 48h migration rate was 38%; the 24h migration rate of the experimental group added with 0.5mg / mL COLI-23 was 44%, and the 48h migration rate was 59%.

[0099] Example 7

[0100] Adhesion effect of recombinant human type II collagen on mouse embryonic fibroblasts NIH3T3 (adhesion experiment verification):

[0101] (1) Mouse embryonic fibroblasts NIH3T3 were digested, centrifuged, resuspended, and diluted to 5×10 4 / mL, 100 μL was inoculated into each well of a 96-well cell culture plate, and then 100 μL of COLII-23 protein solution with a concentration of 1, 5, and 10 mg / mL was added to each well; at the same time, physiological saline was added as a negative control, and cultured at 37°C, 5% CO2, 95% humidity for 6 to 8 hours.

[0102] (2) Discard the supernatant, wash once with PBS, add 100 μL of fresh culture medium and 20 μL of MTT solution to each well, and incubate in an incubator for 4 h at 37°C, 5% CO2, and 95% humidity.

[0103] (3) Aspirate the culture medium, add 150 μL DMSO, and incubate on a shaker for 10 min to allow the crystals to fully dissolve. Select 570 nm (490 nm) as the detection wavelength and 630 nm as the reference wavelength, and measure the light absorbance of each well on an ELISA reader.

[0104] (4) Data analysis: SPSS11.0 was used for statistical analysis. P < 0.05 was considered statistically significant, and P < 0.01 was considered significantly different.

[0105] Test results such as Figure 7 As shown. Figure 7 It can be seen that recombinant human type II collagen COLII-23 has a certain cell adhesion promoting effect on NIH / 3T3 cells.

[0106] Example 8

[0107] Fermentation of recombinant Pichia pastoris (expressing recombinant human type II collagen COLII-23) in a 5L fermenter:

[0108] (1) Seed culture

[0109] Pick a fresh single colony on the YPD plate, inoculate it into a 50 mL centrifuge tube containing 5 mL BMGY medium, and culture it at 30°C, 240 rpm for 18-24 h; transfer it to a 1 L shake flask containing 200 mL BMGY seed medium at a 2% inoculum, and culture it at 30°C, 240 rpm for 14-18 h until the OD 600 = 5 to 6. With an inoculation volume of 10%, 200 mL of seed culture solution was inoculated into a 5 L fermentation tank (containing 2 L of BSM basal salt medium).

[0110] (2) Fermentation culture

[0111] During the glycerol culture stage, the temperature was maintained at 30°C, pH 5.0, the speed was adjusted to 400-800rpm, and DO>20%. When the glycerol in the BSM medium was exhausted, DO = 70-80, and the glycerol addition stage was entered. 50% glycerol was added to further increase the cell density. When the wet weight of the cells was about 180-220g / L, the starvation period began. After starvation for 1 hour, the methanol addition period began. The induction temperature was adjusted to 28°C, and methanol was added to maintain DO at about 20%. Ammonia and phosphoric acid were used to maintain pH throughout the fermentation process. After 60 hours of induction, the tank was released and the supernatant was collected by centrifugation.

[0112] The high-density fermentation of recombinant human type II collagen COLII-23 in a 5L fermenter shows the growth of the bacteria, changes in the wet weight of the bacteria, and the expression curve of the target protein. Figure 8 As shown. Figure 8 It can be seen that induction started at 33 hours of culture and the total culture time was 193 hours; at 60 hours of induction, the highest protein expression level was 785 mg / L.

[0113] Example 9

[0114] Scanning electron microscopy of recombinant human type II collagen COLII-23:

[0115] (1) Sample preparation: Stick conductive glue on the sample table, gently take out the freeze-dried product, stick it on the conductive glue, and perform gold vaporization treatment.

[0116] (2) Observation: The surface structure of the freeze-dried collagen was observed using an electron scanning microscope at magnifications of 300 and 500.

[0117] The results of scanning electron microscopy observation of recombinant human type II collagen COLII-23 are as follows Fig. 9 As shown. Fig. 9 It can be seen that the sample has an obvious porous network structure, which indicates that it has the potential to be applied in the field of biomedical materials.

[0118] In summary, the recombinant human type II collagen fragment COLII-23 of the present invention contains one "tripeptide signal recognition site RGD", two "GER" and three "GEK integrin recognition sites", which not only has excellent effects on promoting cell proliferation and migration in human chondrocytes C28I2, but also shows excellent effects on promoting cell migration and promoting adhesion in human immortalized epidermal cells and mouse embryonic fibroblasts NIH3T3, respectively. It can be widely used in artificial bone, artificial cartilage, artificial cornea, vitreous, wound repair materials, supporting biomaterials, medical cosmetic surgery, cosmetics and other fields.

[0119] 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 human type II collagen fragment, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. The recombinant human type II collagen fragment according to claim 1, characterized in that: The recombinant human type II collagen fragment contains one "tripeptide signal recognition site RGD", two "GER" and three "GEK integrin recognition sites".

3. A gene encoding a recombinant human type II collagen fragment, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1, which is used to encode the recombinant human type II collagen fragment according to claim 1 or 2.

4. The gene encoding the recombinant human type II collagen fragment according to claim 3, characterized in that: The nucleotide sequence shown in SEQ ID NO.1 is a codon-optimized sequence adapted to the Pichia pastoris expression system.

5. An expression vector, characterized in that: Comprising the coding gene of the recombinant human type II collagen fragment as described in claim 3 or 4.

6. The expression vector according to claim 5, characterized in that The expression vector is constructed by adding an EcoRI restriction site sequence to the 5' end of the coding gene sequence and a stop codon and NotI restriction site sequence to the 3' end; then, the sequence fragment is inserted into the pPIC9K expression vector through the EcoRI and NotI restriction sites to obtain the corresponding recombinant expression vector.

7. A recombinant Pichia pastoris engineered bacterium, characterized in that: Comprising the coding gene of the recombinant human type II collagen fragment as described in claim 3 or 4.

8. The recombinant Pichia pastoris engineered bacterium according to claim 7, characterized in that: The construction method of the recombinant Pichia pastoris engineering bacteria is as follows: the expression vector constructed by the coding gene of the recombinant human type II collagen fragment is linearized and then transferred into Pichia pastoris GS115.

9. A method for preparing the recombinant human type II collagen fragment as claimed in claim 1 or 2, characterized in that: Here are the steps: (1) synthesizing the nucleotide sequence encoding the recombinant human type II collagen single chain shown in SEQ ID NO.1; (2) Design PCR primers to amplify and obtain a DNA fragment encoding a single-chain recombinant human type II collagen; (3) constructing a recombinant expression vector comprising the DNA fragment encoding the recombinant human type II collagen single chain, wherein the expression vector is pPIC9K; (4) After linearization, the constructed expression vector was transformed into Pichia pastoris GS115 to obtain recombinant Pichia pastoris engineered bacteria; (5) culturing the above-mentioned recombinant Pichia pastoris to express the recombinant human type II collagen fragment; (6) Separation and purification of the recombinant human type II collagen fragment expressed in step (5): separation and purification of the recombinant type II collagen fragment is performed using dialysis, ion exchange, and secondary dialysis.

10. Use of the recombinant human type II collagen fragment as claimed in claim 1 or 2 in the preparation of artificial bone, artificial cartilage, artificial cornea, vitreous body, wound repair materials and cosmetics.

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