CN118255873A - Recombinant human III type collagen and gene, expression vector, bacteria and application - Google Patents
Recombinant human III type collagen and gene, expression vector, bacteria and application Download PDFInfo
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Abstract
The invention provides recombinant humanized III type collagen and a coding gene thereof, wherein the amino acid sequence is shown as SEQ ID NO.2, the coding gene sequence is shown as SEQ ID NO.1, and the gene sequence is a sequence which is subjected to codon optimization and is suitable for a pichia pastoris expression system. The invention also provides an expression vector comprising the recombinant humanized III type collagen gene and pichia pastoris engineering bacteria. Meanwhile, the invention also provides preparation and application of the recombinant humanized III-type collagen. The recombinant human type III collagen has small molecular weight and excellent activity, has excellent cell proliferation promoting effect in mouse embryo fibroblast and cell proliferation and migration promoting effect in human immortalized epidermal cell, and may be used widely in wound repairing material, medicine, cosmetics, health product, etc.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to recombinant human type III collagen, a gene, an expression vector, bacteria and application.
Background
Collagen is the most abundant structural protein in the human body, has special biological functions, and provides important mechanical properties including elasticity, toughness and the like in tissues. Accordingly, collagen-related biomaterials are commonly used for tissue repair and regeneration. In recent years, the market demand for collagen has been increasing due to the development of socioeconomic performance. Most initial extraction methods of collagen are extracted from animal tissues by acid or alkali hydrolysis, and heterologous extraction methods have obvious defects, and have strong immune rejection, potential safety hazards of viruses, infectious diseases and the like. The human collagen has weak antigenicity, can induce cell proliferation and migration and promote cell adhesion, and is an important and safe biological material.
Type III human collagen is a fibrillar collagen secreted by fibroblasts and mesenchymal cells, a homotrimer formed by three alpha 1 (III) chains (COL 3A 1). In pathological aspects, type III collagen is associated with body inflammation, such as lung injury, viral and non-viral liver disease, kidney fibrosis, hernia, vascular related diseases, and the like. Mutations in type III collagen can lead to congenital connective tissue hypoplastic syndrome, vascular defects, and aortic and aneurysms. In the field of skin care, the content of type III collagen in skin gradually decreases with age, so that various cosmetics, medical and aesthetic products and the like can achieve the aims of moisturizing and removing wrinkles by supplementing collagen to skin. Type III collagen has many potential applications in the market, such as artificial cornea, artificial heart valve, endometrial repair made from type III collagen; the III type collagen is used as a medicine, and can promote cancer cells to enter and maintain a dormant state and inhibit tumor proliferation by adding the III type collagen into cancer tissues. According to the above, type III collagen has a huge market of application.
However, although type III collagen has a huge application market, the related research on the high-activity collagen fragments is not enough at present.
With the development of genetic engineering techniques, expression systems based on animals, plants and microorganisms have been gradually established, and among them, the studies of microbial expression systems have been most widely conducted. Li Jia and the like, a bacillus expression system is used, and a human III type collagen peptide (China patent No. CN 114539390B) containing a non-helical region N section, a non-helical region C section and 100% of human collagen fragments is obtained through splicing recombination, but the bacillus expression system has the defects of low transcription level of a promoter, easy decomposition of an expression product, toxicity of a required inducer, high price and the like. Yang Xia et al expressed a human collagen type III peptide (China patent No. CN 103122027B) using E.coli, fan Dai subject group expressed a type III collagen (China patent No. CN 115991763A) by E.coli, but E.coli expression system still faced some difficult problems at present, and the produced collagen had endotoxin, had complicated purification steps, lacked post-translational modification, and increased production cost. In contrast, pichia pastoris expression systems are increasingly being used for expressing recombinant collagen with high activity due to their high yield, no endotoxin, simple purification means, low cost, and possibility of post-translational modification. Thus, attempts may be made to construct a pichia pastoris-based human type III collagen expression system.
Meanwhile, integrin is a transmembrane receptor, and heterodimers formed by an alpha subunit and a beta subunit can mediate signal transmission in the intracellular and extracellular environments and rapidly and sensitively respond to changes in the environments. Thus, integrins are involved in cell signaling, cell cycle regulation, cell morphology, and cell movement, in addition to mechanical action across the membrane. The amino acid sequence of the integrin recognition site comprises GER, GEK and the like, and collagen fragments which contain the corresponding amino acid sequences and are exposed at the site can be recognized by the integrin on the cell membrane, and the cell-related efficacy is promoted through signal transduction. Therefore, on the basis of the human-derived type III collagen, the recombinant human-derived type III collagen with more excellent biological activity can be obtained through sequence design and optimization based on the integrin recognition site.
Disclosure of Invention
The invention aims to solve the technical problems of providing a recombinant human type III collagen, a gene, an expression vector, bacteria and application, wherein the obtained recombinant human type III collagen fragment has small molecular weight and excellent activity, has excellent cell proliferation promoting effect in mouse embryo fibroblasts and also has excellent cell proliferation and migration promoting effect in human immortalized epidermal cells, and can be widely applied to the fields of wound repair materials, medical cosmetology, cosmetics, health care products and the like.
The invention adopts the following technical scheme to solve the technical problems:
A recombinant human type III collagen has an amino acid sequence shown in SEQ ID NO. 2.
The recombinant human-derived type III collagen fragment provided by the invention has small molecular weight, contains two GER and one GEK integrin recognition site, has excellent activity, has excellent cell proliferation promoting effect in mouse embryo fibroblasts and also has excellent cell proliferation and migration promoting effect in human immortalized epidermal cells, and can be widely applied to the fields of wound repair materials, medical cosmetology, cosmetics, health care products and the like.
A recombinant human-derived type III collagen encoding gene, wherein the gene encodes the recombinant human-derived type III collagen according to claim 1, and the nucleotide sequence is shown as SEQ ID NO. 1.
As one of the preferable modes of the invention, the nucleotide sequence shown in SEQ ID NO.1 is a sequence which is subjected to codon optimization and is suitable for a Pichia pastoris expression system.
As one of the preferred modes of the invention, the online codon optimization software is adopted to change the corresponding original recombinant human type III collagen codon base into the base favored by pichia pastoris codon without changing the protein coding sequence.
An expression vector comprising the recombinant human type III collagen encoding gene.
As one of the preferable modes of the invention, the construction method of the expression vector comprises the following steps: an EcoRI enzyme cutting site sequence is added at the 5 'end of the coding gene sequence, and a stop codon and a NotI enzyme cutting site sequence are added at the 3' end; then, the sequence fragment is inserted into the pPIC9K expression vector through EcoRI and NotI restriction sites to obtain a corresponding recombinant expression vector.
A recombinant Pichia pastoris engineering bacterium comprises the recombinant human source III type collagen encoding gene.
As one of preferred embodiments of the present invention, the construction method of the engineering bacterium is as follows: and linearizing an expression vector constructed by the recombinant humanized III type collagen encoding gene, and transferring into Pichia pastoris GS115.
The preparation method of the recombinant humanized III type collagen comprises the following steps:
(1) Synthesizing a nucleotide sequence shown in SEQ ID NO.1 for encoding a recombinant human source III type collagen single chain;
(2) Designing PCR primers, and amplifying DNA fragments encoding recombinant human source III type collagen single chains, wherein the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4;
(3) Constructing a recombinant expression vector comprising the DNA fragment for encoding the recombinant humanized III type collagen single chain, wherein the expression vector is pPIC9K;
(4) Linearizing the constructed expression vector, and transferring into Pichia pastoris GS115 to obtain recombinant Pichia pastoris engineering bacteria;
(5) Culturing the recombinant pichia pastoris engineering bacteria and expressing recombinant human III type collagen fragments;
(6) Isolating and purifying the recombinant human type III collagen fragment expressed in the step (5): recombinant type III collagen was isolated and purified using dialysis (dialysate: 10mM sodium acetate, pH 4.5), ion exchange, and secondary dialysis (dialysate: PBS, pH 7.0).
The recombinant human-derived type III collagen can be applied to the fields of wound repair materials, medical cosmetology and plastic, cosmetics and health care products.
Compared with the prior art, the invention has the advantages that:
(1) The recombinant human source III type collagen fragment provided by the invention has small molecular weight, contains two GER and one GEK integrin recognition site (the GER and the GEK site can be recognized by the integrin recognition site on a cell membrane and then transferred to cells through signal transduction, so that cell proliferation and adhesion are promoted), has excellent activity, has excellent cell proliferation promoting effect in mouse embryo fibroblasts, has excellent cell proliferation and migration promoting effect in human immortalized epidermal cells, can be widely applied to the fields of wound repair materials, medical cosmetology, cosmetics, health care products and the like, and has good application prospect especially in skin injury;
(2) According to the expression characteristics of pichia pastoris, the nucleotide sequence for encoding the recombinant human source III type collagen is also subjected to codon optimization, so that the nucleotide sequence for encoding the recombinant human source III type collagen is more suitable for an expression system of pichia pastoris, and a recombinant human source III type collagen high-yield strain is finally obtained through liquid small test expression screening;
(3) The invention can obtain high recovery rate and high purity recombinant human source III type collagen by combining dialysis and ion exchange chromatography for purification;
(4) The invention uses pichia pastoris as a host to produce recombinant human source III type collagen, which can fully support the biological functions of collagen products produced by the collagen;
(5) The pPIC9K used in the invention is an expression vector, can continuously secrete and express collagen in the life activities of engineering bacteria, has simple fermentation process and less impurity protein, and provides an advantageous strain for large-scale fermentation production.
Drawings
FIG. 1 is a diagram showing the active sites of GER and GEK in the amino acid sequence of recombinant human type III collagen COLIII-20 of example 1;
FIG. 2 is an agarose gel electrophoresis chart of a DNA fragment of recombinant human type III collagen COLIII-20 in example 1 (in the figure, M is marker, lanes 1 and 2 are samples; the target band size is 627 bp);
FIG. 3 is an electrophoresis chart of a protein obtained by separating and purifying recombinant human type III collagen COLIII-20 in example 4 (in the figure, lane 1 is a molecular weight Marker, lanes 2,3, and 4 are hetero proteins, lane 5 is a target protein; the electrophoresis detection molecular weight of COLIII-20 protein is about 20kDa, corresponding to a protein comprising the amino acid sequence of SEQ ID NO. 2);
FIG. 4 is a graph showing the effect of recombinant human type III collagen COLIII-20 on proliferation of mouse embryonic fibroblasts and human immortalized epidermal cells in example 5 (in the figure, panel A shows the effect on mouse embryonic fibroblasts NIH/3T3, and panel B shows the effect on human immortalized epidermal cells HaCaT);
FIG. 5 is a graph showing the effect of recombinant human type III collagen COLIII-20 on the mobility of human immortalized epidermal cells in example 5;
FIG. 6 is a graph showing the growth of recombinant human type III collagen COLIII-20 in a 5L fermenter and the expression level of the target protein in example 6.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
The Pichia pastoris GS115 strain and the expression vector pPIC9K selected in the following examples are available strains and vectors. The medium formulation used was as follows:
1) YPD complete medium:
To 900mL of water was added 10g/L of yeast extract, 20g/L of peptone, and 100mL of a sterile 10 Xglucose solution after autoclaving. The solid medium contained 1.5% agar.
2) MD medium:
To prepare 1L, 800mL of water was autoclaved, cooled to 60℃and then 100mL 10X YNB,2mL 500X mL of 10 Xglucose was added. The solid medium contained 2% agar.
3) BMGY medium:
To 700mL of water, 10g/L of yeast extract, 20g/L of peptone, and 100mL of 10 Xglycerol were added, followed by 1L of 1M potassium phosphate buffer (pH 6.0), 100mL 10X YNB,2mL 500X biotin, and 100mL of 10 Xglycerol, after which the mixture was autoclaved and cooled to room temperature.
4) BMMY medium:
to 700mL of water, 10g/L of yeast extract, 20g/L of peptone, and 100mL of 10 Xmethanol were added, followed by 1L of 1M potassium phosphate buffer (pH 6.0), 100mL 10X YNB,2mL 500X biotin, and 100mL of 10 Xmethanol, after which the mixture was autoclaved and cooled to room temperature.
5) BSM medium:
CaSO 4 0.93g/L,K2SO4 18.2g/L,MgSO4·7H2 O14.9 g/L, KOH 4.13g L, glycerin 40g/L, defoamer 1g/L, and 85% phosphoric acid 26.7mL after sterilization.
6) PTM1 trace elements:
CuSO4·5H2O 6g/L,NaI 0.08g/L,MnSO4·H2O 3g/L,NaMoO4·2H2O 0.2g/L,H3BO30.02g/L,CoCl2 0.5g/L,ZnCl2 20g/L,FeSO4·7H2O 65g/L, Biotin 0.2g/L, H 2SO4 mL.
Meanwhile, DMEM basal medium, PBS, fetal Bovine Serum (FBS), pancreatin (vivacell) and calf serum (BCS), mouse embryo fibroblast line NIH/3T3 and human immortalized epidermal cell line HaCaT (Oricell) were used in the following examples.
Example 1
Recombinant human source III type collagen COLIII-20 coding gene:
The final screening of the optimized recombinant human III-type collagen COLIII-20 coding gene in this example has the nucleotide sequence shown in SEQ ID NO.1 and the corresponding amino acid sequence shown in SEQ ID NO.2 (GER and GEK active sites in the amino acid sequence of recombinant human III-type collagen COLIII-20 are shown in FIG. 1).
Gene screening optimization process:
(1) Based on the human source III type collagen gene sequence in GeneBank, an original sequence fragment containing two GER and one GEK integrin recognition site sequences is found, and is shown as SEQ ID NO. 3.
(2) Aiming AT the codon preference of pichia pastoris and the AT content of the DNA sequence, online codon optimization software is adopted to change the corresponding original recombinant humanized III type collagen codon base into the base favored by the pichia pastoris codon without changing the protein coding sequence, and the optimized gene sequence is shown as SEQ ID NO. 1; the optimized gene sequence is synthesized by Shanghai biological engineering Co.
Cloning of the genes:
Specific primers were designed and PCR was used to amplify DNA fragments encoding single strands of recombinant human type III collagen COLIII-20, the PCR system being shown in Table 1.
TABLE 1PCR amplification reaction System and reaction conditions
The target fragment was detected by 1% agarose gel electrophoresis, and the result is shown in FIG. 2 (target band size: 627 bp).
Example 2
Construction of recombinant human-derived type III collagen COLIII-20 expression vector:
An EcoRI enzyme cutting site sequence is added at the 5 'end of the coding gene sequence, and a stop codon and a NotI enzyme cutting site sequence are added at the 3' end; then, the sequence fragment is inserted into the pPIC9K expression vector through EcoRI and NotI restriction sites, and the corresponding recombinant expression vector pPIC9K-COLIII-20 is obtained.
Example 3
Construction of recombinant pichia pastoris engineering bacteria:
(1) And linearizing the successfully constructed recombinant expression vector plasmid pPIC9K-COLIII-20, and transferring the linearized recombinant expression vector plasmid into the competence of Pichia pastoris GS 115.
(2) The monoclonal colony is selected from the transformed MD plate and cultured in a 48-hole culture plate containing 500 mu L of YPD (0.75 mg/mL G418) culture medium at 30 ℃ and 240rpm for 18-24 hours; sequentially transferring to YPD liquid culture medium with gradually increased G418 content for culturing.
(3) The strain with good growth vigor is fermented, and the steps are as follows:
Inoculating the bacterial liquid into 10mL/50mL BMGY with an inoculum size of 2%, and culturing at 30 ℃ and 240rpm for 18-24 h;
Centrifuging, discarding supernatant, adding 10mL BMMY for resuspension, culturing at 28 ℃ and 240rpm for 48h, and adding 100% methanol every 24h to reach a final concentration of 1.5%;
Centrifuging, and taking the supernatant to carry out SDS-PAGE analysis to obtain recombinant pichia pastoris engineering bacteria with high expression quantity.
Example 4
Production and preparation of recombinant human III type collagen COLIII-20 and purification:
(1) Activating recombinant Pichia pastoris engineering bacteria (glycerol strain) successfully constructed in YPD (G418 with corresponding resistance) culture medium at 30 ℃ at 240rpm for 18h;
(2) Transferring 2% of inoculation amount into 200mL/1L BMGY, culturing at 30 ℃ and 240rpm for 26-28 h, and completely consuming glycerol in a culture medium;
(3) Taking 200mL BMMY re-suspension thalli, and culturing for 48h at 25 ℃ and 240 rpm; 100% methanol was added every 24h to a final concentration of 1.5%.
(4) Filtering the supernatant after fermentation with 0.22 μm water-based filter membrane to obtain clear fermentation supernatant, and separating and purifying recombinant type III collagen COLIII-20 by dialysis (dialysate: 10mM sodium acetate, pH 4.5), ion exchange chromatography, and secondary dialysis (dialysate: PBS, pH 7.0).
FIG. 3 is an electrophoresis chart of a protein obtained by separating and purifying recombinant human type III collagen COLIII-20. The separated and purified COLIII-20 protein liquid is analyzed by gel filtration chromatography and non-reducing-SDS-PAGE, the purity is more than 95%, and the recovery rate is more than 85%.
Example 5
Biological activity detection of recombinant human type III collagen COLIII-20:
1. Measurement of recombinant human type III collagen by MTT method promotes proliferation of "mouse embryo fibroblasts" and "human immortalized epidermal cells":
(1) Taking mouse embryo fibroblast NIH/3T3 or human immortalized epidermal cell HaCaT, digesting, centrifuging, re-suspending, diluting to 3X 10 4/mL, and inoculating 100 mu L to 96-well cell culture plate in each well; cell-free medium was also inoculated as a blank, incubated at 37℃with 5% CO 2 at 95% humidity for 24h.
(2) COLIII-20 protein suspensions 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL, 0.015625mg/mL were formulated.
(3) The medium was discarded, the blank medium, the negative medium, the positive medium with 5% DMSO, the experimental group with medium with different concentrations COLIII-20 protein solution, 37℃and 5% CO 2, 95% humidity were added, and the culture was performed for 24 hours.
(4) Mu.L MTT was added to each well and incubated in an incubator for 4h at 37℃with 5% CO 2 at 95% humidity. 150 μl DMSO was added and incubated for 10min with shaking to allow the crystals to dissolve well, and the absorbance at 490nm was measured with an ELISA.
The detection results are shown in FIG. 4. As can be seen from fig. 4A, B: taking the negative control group as 100%, the survival rate of the NIH/3T3 experimental group added with COLIII-20 is 124%; the survival rate of the HaCaT experimental group added with COLIII-20 is 135%.
2. Scratch experiments measure that recombinant human type III collagen promotes migration of 'human immortalized epidermal cells':
(1) Cell culture: a Marker pen was used to draw 5 lines transversely per well behind the 6-well plate, with each line spaced about 0.5cm apart as a Marker. HaCaT cells are selected, a blank group, a control group and an experiment group are arranged, after digestion, centrifugation and re-suspension, the cells are diluted to 2X 10 6 cells/mL, 2mL of cell suspension is used for each hole, the cell density of each group is ensured to be the same, and the cells can reach a confluence state of 95-100% after being cultured for 24 hours.
(2) Scratch experiment: after cell culture for 24h, the cell layer was scratched vertically against the plate plane with a 200. Mu.L gun head, the cells were rinsed 3 times with PBS, and the scratched cells were removed, and 2mL of COLIII-20 protein-containing medium was added to 6 wells at final concentrations of 0.05mg/mL, 0.005mg/mL, and 0.0005mg/mL, respectively. The blank group was added with medium only. The control group was added with commercially available recombinant collagen.
(3) Culturing at 37deg.C, 5% CO 2, and 95% humidity in incubator, and performing microscopic photographing at fixed position after 0 hr, 24 hr, and 48 hr.
(4) Scratch area was measured using Image J: and processing scratch pictures (0 h, 24h/48 h) by using software to finally obtain the mobility of the cells.
The detection results are shown in FIG. 5. As can be seen from fig. 5A, B: blank (NC) 24h mobility was 10% and 48h mobility was 13%; the 24h mobility of the commercial recombinant collagen is 17%, and the 48h mobility is 20%; the experimental group to which 0.0005mg/mLCOLIII-20 was added had a 24h mobility of 25% and a 48h mobility of 37%.
Example 6
Fermenting the recombinant Pichia pastoris engineering bacteria (expressing COLIII-20 protein) in a 5L fermentation tank:
(1) Seed culture
Picking a single colony of fresh Pichia pastoris engineering bacteria on a YPD plate, and inoculating the single colony into a50 mL centrifuge tube containing 5mL BMGY culture medium, and culturing at 30 ℃ and 240rpm for 18-24 h; transfer to 1L shake flask containing 200mL BMGY seed medium at 30℃and 240rpm for 14-18 h to OD 600 = 5-6 at 2% inoculum size. 200mL of seed culture was all placed in a 5L fermenter (containing 2L of BSM basal salt medium) at an inoculum size of 10%.
(2) Fermentation culture
The glycerol culture stage maintains the temperature at 30 ℃, the pH value is 5.0, the rotating speed is regulated to 400-800 rpm, and DO is maintained to be more than 20%. When the glycerol of the BSM culture medium is exhausted, DO=70-80 enters a glycerol feeding stage, 50% glycerol starts to be fed in, and the cell density is further increased. When the wet weight of the thalli is about 180-220 g/L, the thalli enters a starvation period, after starvation for 1h, the thalli enters a methanol feeding period, the induction temperature is regulated to 26 ℃, and methanol starts to be fed, so that DO is maintained at about 20%. The pH of the whole fermentation process is maintained by ammonia and phosphoric acid. And (5) inducing for 54 hours, placing the mixture in a tank, and centrifuging to collect supernatant.
COLIII-20 the growth of the fermentation cells and the expression level of the target protein in a 5L fermenter are shown in FIG. 6. As can be seen from fig. 6: when the induction is carried out for 24 hours, the yield of COLIII-20 is up to 0.67g/L; when the induction is carried out for 54 hours, the yield of COLIII-20 is 0.97g/L at maximum.
In conclusion, the recombinant human type III collagen COLIII-20 has small molecular weight, contains two GERs and one GEK integrin recognition site, has excellent activity, has excellent effect of promoting cell proliferation in mouse embryo fibroblasts and excellent effect of promoting cell proliferation and migration in human immortalized epidermal cells, can be widely applied to the fields of wound repair materials, medical cosmetology and plastic, cosmetics, health care products and the like, and has good application prospect especially in skin injury.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A recombinant human-derived type III collagen is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
2. A recombinant human-derived type III collagen encoding gene is characterized in that the gene encodes the recombinant human-derived type III collagen according to claim 1, and the nucleotide sequence is shown as SEQ ID NO. 1.
3. The recombinant human-derived type III collagen encoding gene according to claim 2, wherein the nucleotide sequence of SEQ ID No.1 is codon optimized to adapt to the sequence of the pichia pastoris expression system.
4. The recombinant human type III collagen encoding gene according to claim 3, wherein the corresponding original recombinant human type III collagen codon base is changed to a Pichia pastoris codon preferred base without changing the protein encoding sequence by using on-line codon optimization software.
5. An expression vector comprising the recombinant human type III collagen encoding gene according to any one of claims 2 to 4.
6. The expression vector of claim 5, wherein the expression vector is constructed by the following steps: an EcoRI enzyme cutting site sequence is added at the 5 'end of the coding gene sequence, and a stop codon and a NotI enzyme cutting site sequence are added at the 3' end; then, the sequence fragment is inserted into the pPIC9K expression vector through EcoRI and NotI restriction sites to obtain a corresponding recombinant expression vector.
7. A recombinant pichia pastoris engineering bacterium, comprising the recombinant human type III collagen encoding gene according to any one of claims 2 to 4.
8. The engineering bacterium according to claim 7, wherein the construction method of the engineering bacterium comprises the following steps: and linearizing an expression vector constructed by the recombinant humanized III type collagen encoding gene, and transferring into Pichia pastoris GS115.
9. A method of preparing recombinant human type III collagen according to claim 1, comprising the steps of:
(1) Synthesizing a nucleotide sequence shown in SEQ ID NO.1 for encoding a recombinant human source III type collagen single chain;
(2) Designing PCR primers, and amplifying DNA fragments encoding recombinant human source III type collagen single chains, wherein the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4;
(3) Constructing a recombinant expression vector comprising the DNA fragment for encoding the recombinant humanized III type collagen single chain, wherein the expression vector is pPIC9K;
(4) Linearizing the constructed expression vector, and transferring into Pichia pastoris GS115 to obtain recombinant Pichia pastoris engineering bacteria;
(5) Culturing the recombinant pichia pastoris engineering bacteria and expressing recombinant human III type collagen fragments;
(6) Isolating and purifying the recombinant human type III collagen fragment expressed in the step (5): recombinant type III collagen was isolated and purified using dialysis, ion exchange, and secondary dialysis.
10. Use of the recombinant human-derived type III collagen according to claim 1 in the field of wound repair materials, medical cosmetology, cosmetics, health care products.
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