CN114644707A - Preparation method of gene recombinant collagen oligopeptide - Google Patents
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 59
- 108010035532 Collagen Proteins 0.000 title claims abstract description 59
- 229920001436 collagen Polymers 0.000 title claims abstract description 59
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 41
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 41
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 29
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 29
- 101100425522 Caenorhabditis elegans mys-1 gene Proteins 0.000 claims abstract description 27
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 15
- 239000013612 plasmid Substances 0.000 claims abstract description 14
- 229920001184 polypeptide Polymers 0.000 claims abstract description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 12
- 101150067498 mys-1 gene Proteins 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 238000005215 recombination Methods 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 7
- 230000006798 recombination Effects 0.000 claims abstract description 7
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- 241000588724 Escherichia coli Species 0.000 claims abstract description 4
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 239000003480 eluent Substances 0.000 claims abstract description 4
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- 230000001131 transforming effect Effects 0.000 claims abstract description 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 42
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 30
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 15
- 229910052759 nickel Inorganic materials 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 7
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 7
- 235000019800 disodium phosphate Nutrition 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 102100029727 Enteropeptidase Human genes 0.000 claims description 6
- 108010013369 Enteropeptidase Proteins 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 230000000149 penetrating effect Effects 0.000 claims description 6
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- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 claims description 6
- 239000011534 wash buffer Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000002860 competitive effect Effects 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
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- 239000012149 elution buffer Substances 0.000 claims description 3
- 230000031700 light absorption Effects 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 230000001172 regenerating effect Effects 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 239000012264 purified product Substances 0.000 claims 1
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 10
- 230000032683 aging Effects 0.000 abstract description 8
- 230000008439 repair process Effects 0.000 abstract description 6
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- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 210000003491 skin Anatomy 0.000 description 9
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- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- 241000700605 Viruses Species 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a preparation method of gene recombinant collagen oligopeptide. The preparation method of the gene recombinant collagen oligopeptide comprises the following steps: s1, synthesizing MYS-1 gene by PCR reaction with a pair of complementary primers; s2, carrying out double enzyme digestion on the MYS-1 gene to obtain a recombinant plasmid; s3, transforming and expressing host escherichia coli by using the recombinant plasmid to obtain expression engineering bacteria; s4, inducing expression engineering bacteria to express the fusion protein; s5, purification: collecting the eluent; s6, enzyme digestion of the fusion protein and purification of the target polypeptide MYS-1; s7, purifying and preparing the collagen oligopeptide MYS-1 by using a high performance liquid chromatography technology to obtain the high-purity gene recombination high-stability collagen oligopeptide MYS-1. The length of the collagen oligopeptide prepared by the invention is far shorter than that of natural human collagen and related hydrolyzed polypeptide, the molecular weight is small, the preparation is easy, the efficiency is high, and the collagen oligopeptide has good biological activities of oxidation resistance, aging resistance, repair promotion, melanin generation inhibition and the like.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a preparation method of gene recombination collagen oligopeptide.
Background
Collagen is a high molecular functional protein, which is the main component of skin and accounts for 80% of the proportion of the dermis layer of the skin, and forms a fine elastic net in the skin, firmly locks water and supports the skin.
With the aging, the collagen is gradually lost, so that the peptide bonds and the elastic nets of the collagen supporting the skin are broken, the spiral net structure is damaged, the skin tissue is oxidized, atrophied and collapsed, and the skin can have aging phenomena of dryness, wrinkles, looseness, inelasticity and the like, so that the collagen supplement for women is necessary for delaying aging. Parent blautid assertion of collagen: "fundamentally, the skin aging process is the process of collagen loss". After long-term administration of LOC collagen, people can repair broken and aged fiber elastic nets and stabilize the structural shape of the fiber elastic nets, and can own a dermis reservoir. When the collagen fiber elastic net of the real cortex is oxidized and broken, the supporting effect on the epidermis disappears, and the water cannot be locked and stored; thus causing uneven collapse and wrinkles are generated.
At present, the production of collagen mainly comprises the traditional extraction, chemical synthesis and gene recombination preparation technology. The traditional extraction technology obtains a series of hydrolysates by hydrolyzing natural collagen, but the application risk of immunogenicity is difficult to avoid; the cost of chemically synthesizing collagen is high; the gene recombination technology has the advantages of high yield, low cost, high activity of produced protein, polypeptide or oligopeptide, no virus hidden danger and the like, so that the gene recombination micromolecule collagen polypeptide has good industrialization prospect and huge market application potential.
Disclosure of Invention
The invention aims to provide a preparation method of gene recombinant collagen oligopeptide with an anti-aging effect.
In order to achieve the purpose, the invention adopts the following technical scheme that the preparation method of the gene recombinant collagen oligopeptide comprises the following steps:
s1, synthesizing MYS-1 gene by PCR reaction with a pair of complementary primers;
s2, carrying out double enzyme digestion on the plasmid pET32a and the MYS-1 gene prepared in the step S1 by using KpnI enzyme and XhoI enzyme respectively, and then carrying out double enzyme digestion on the MYS-1 gene and the plasmid pET32a to obtain a recombinant plasmid pET32 a-MYS-1;
s3, transforming and expressing host escherichia coli E.coli BL21(DE3) by using a recombinant plasmid pET32a-MYS-1 to obtain expression engineering bacteria pET32a-MYS-1/BL21(DE 3);
s4, inducing expression engineering bacteria pET32a-MYS-1/BL21(DE3) to express fusion protein consisting of Trx tag protein, His tag protein, collagen oligopeptide MYS-1 and the like;
s5, purifying the expressed His-tagged fusion protein by using a nickel column: after the protein is loaded, the fusion protein with the His label is specifically combined with the nickel column, and other hybrid proteins flow out because the other hybrid proteins are not specifically combined with the nickel column; gradient elution is carried out by imidazole, and the imidazole and Ni2+ are combined with a nickel column in a competitive way so as to release fusion protein and collect eluent;
s6, enzyme digestion of the fusion protein and purification of the target polypeptide MYS-1;
s7, purifying and preparing collagen oligopeptide MYS-1 by using a high performance liquid chromatography technology to obtain high-purity gene recombination high-stability collagen oligopeptide MYS-1;
wherein the primers in step S1 are:
primer 1:
primer 2:
GGTACCis a KpnI restriction enzyme cutting site,CTCGAGis XhoI restriction site;
the conditions of the PCR reaction in step S1 were: hold 95 ℃ for 5 minutes, perform 30 temperature cycles of 1 minute 95 ℃ to 1 minute 50 ℃ and then 1 minute 70 ℃ and finally hold 70 ℃ for 10 minutes.
In some embodiments, step S5, a gentle buffer and a washing buffer are used, and the gentle buffer comprises: 50mM Na2HPO4, 0.3M NaCl, pH 8.0;
the wash buffer included the following components: 50mM Na2HPO4, 0.3M NaCl, 10-50 mM imidazole, pH 8.0.
In some embodiments, the composition of the solution for eluting the nickel column in step S5 is preferably as follows: 50mM Na2HPO4, 0.3M NaCl, 250mM imidazole, pH 8.0.
In some embodiments, in step S6, the steps of cleavage of the fusion protein and purification of the polypeptide of interest MYS-1 are as follows:
(1) dialyzing the fusion protein eluate in 1 XPBS (pH7.6) buffer solution, adding enterokinase (EK, 5 IU/. mu.L) into the dialyzed fusion protein solution, wherein 5U enterokinase is added into every 500. mu.g of fusion protein, and the enzyme digestion conditions are as follows: enzyme digestion is carried out for 6h at the temperature of 25 ℃;
(2) and (3) passing the solution system after enzyme digestion through a balanced Ni-NTA purification column, collecting penetrating liquid, eluting the label protein and other combined hybrid proteins on the column by using an elution buffer solution containing 250mmol/L imidazole, wherein the total elution volume is 5 times of that of the column bed, regenerating the Ni-NTA purification column, and collecting the penetrating liquid which is a primary purification product containing the collagen oligopeptide MYS-1.
In some embodiments, in step S7, the step of purifying the collagen oligopeptide MYS-1 by high performance liquid chromatography is as follows:
A. the mobile phase A is obtained by adding trifluoroacetic acid (TFA) to 100% by volume of acetonitrile, and the final concentration of TFA is 0.1% by volume; mobile phase B trifluoroacetic acid (TFA) was added to 100% water at a final TFA concentration of 0.1% by volume; the flow rate is 1.0mL/min, and the elution is carried out in a linear gradient manner for 20 min;
B. collecting the elution peak of the collagen oligopeptide MYS-1 from 55-80% (v/v) of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 218 nm; and carrying out mass spectrum identification.
The invention has the beneficial effects that: according to the preparation method of the gene recombinant collagen oligopeptide, the length of the prepared collagen oligopeptide is far shorter than that of natural human collagen and related hydrolyzed polypeptides, the molecular weight is small, the preparation is easy, and the efficiency is high; compared with natural collagen or collagen-like peptide from animal sources, the collagen or collagen-like peptide has better biocompatibility and biological safety, has good biological activities of oxidation resistance, aging resistance, promotion of repair, inhibition of melanin generation and the like, can remarkably promote the oxidative damage repair of skin, reduce the melanin generation and resist aging, and has no obvious toxic or side effect; can be used in the fields of food, health products, cosmetics and the like, has wide application range and good industrial application value.
Drawings
FIG. 1 is a schematic flow chart of the method for preparing the gene recombinant collagen oligopeptide of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Examples
As shown in FIG. 1, the preparation method of the gene recombinant collagen oligopeptide comprises the following steps:
s1, synthesizing MYS-1 gene by PCR reaction with a pair of complementary primers;
s2, carrying out double enzyme digestion on the plasmid pET32a and the MYS-1 gene prepared in the step S1 by using KpnI enzyme and XhoI enzyme respectively, and then carrying out double enzyme digestion on the MYS-1 gene and the plasmid pET32a to obtain a recombinant plasmid pET32 a-MYS-1;
s3, transforming and expressing host escherichia coli E.coli BL21(DE3) by using a recombinant plasmid pET32a-MYS-1 to obtain expression engineering bacteria pET32a-MYS-1/BL21(DE 3);
s4, inducing expression engineering bacteria pET32a-MYS-1/BL21(DE3) to express fusion protein comprising Trx tag protein, His tag protein, collagen oligopeptide MYS-1 and the like;
s5, purifying the expressed His-tagged fusion protein by using a nickel column: after the protein is loaded, the fusion protein with the His label is specifically combined with the nickel column, and other hybrid proteins flow out because of not being specifically combined with the nickel column; gradient elution is carried out by imidazole, and the imidazole and Ni2+ are combined with a nickel column in a competitive way so as to release fusion protein and collect eluent;
s6, carrying out enzyme digestion on the fusion protein and purifying the target polypeptide MYS-1;
s7, purifying and preparing collagen oligopeptide MYS-1 by using a high performance liquid chromatography technology to obtain high-purity gene recombination high-stability collagen oligopeptide MYS-1;
wherein the primers in step S1 are:
primer 1:
primer 2:
GGTACCis a KpnI restriction enzyme cutting site,CTCGAGis XhoI restriction site;
the conditions of the PCR reaction in step S1 are: hold 95 ℃ for 5 minutes, perform 30 temperature cycles of 1 minute 95 ℃ to 1 minute 50 ℃ and then 1 minute 70 ℃ and finally hold 70 ℃ for 10 minutes.
In step S5, a gentle buffer and a washing buffer are used, and the gentle buffer comprises: 50mM Na2HPO4, 0.3M NaCl, pH 8.0;
the wash buffer included the following components: 50mM Na2HPO4, 0.3M NaCl, 10-50 mM imidazole, pH 8.0.
In step S5, the composition of the solution for eluting the nickel column is preferably as follows: 50mM Na2HPO4, 0.3m naci, 250mM imidazole, pH 8.0.
In step S6, the steps of enzyme digestion of the fusion protein and purification of the target polypeptide MYS-1 are as follows:
(1) dialyzing the fusion protein eluate in 1 XPBS (pH7.6) buffer solution, adding enterokinase (EK, 5 IU/. mu.L) into the dialyzed fusion protein solution, wherein 5U enterokinase is added into every 500. mu.g of fusion protein, and the enzyme digestion conditions are as follows: enzyme digestion is carried out for 6h at the temperature of 25 ℃;
(2) and (3) passing the solution system after enzyme digestion through a balanced Ni-NTA purification column, collecting penetrating liquid, eluting the label protein and other combined hybrid proteins on the column by using an elution buffer solution containing 250mmol/L imidazole, wherein the total elution volume is 5 times of that of the column bed, regenerating the Ni-NTA purification column, and collecting the penetrating liquid which is a primary purification product containing the collagen oligopeptide MYS-1.
In step S7, the purification of collagen oligopeptide MYS-1 by high performance liquid chromatography comprises the following steps:
A. the mobile phase A is obtained by adding trifluoroacetic acid (TFA) to 100% by volume of acetonitrile, and the final concentration of TFA is 0.1% by volume; mobile phase B trifluoroacetic acid (TFA) was added to 100% water at a final TFA concentration of 0.1% by volume; the flow rate is 1.0mL/min, and the elution is carried out in a linear gradient manner for 20 min;
B. collecting the elution peak of the collagen oligopeptide MYS-1 from 55-80% (v/v) of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 218 nm; and carrying out mass spectrum identification.
The invention has the beneficial effects that: according to the preparation method of the gene recombinant collagen oligopeptide, the length of the prepared collagen oligopeptide is far smaller than that of natural human collagen and related hydrolyzed polypeptides, the molecular weight is small, the preparation is easy, and the efficiency is high; compared with natural collagen or collagen-like peptide from animal sources, the collagen or collagen-like peptide has better biocompatibility and biological safety, has good biological activities of oxidation resistance, aging resistance, promotion of repair, inhibition of melanin generation and the like, can remarkably promote the oxidative damage repair of skin, reduce the melanin generation and resist aging, and has no obvious toxic or side effect; can be used in the fields of food, health products, cosmetics and the like, has wide application range and good industrial application value.
What has been described above are merely some of the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (5)
1. The preparation method of the gene recombinant collagen oligopeptide comprises the following steps:
s1, synthesizing MYS-1 gene by PCR reaction with a pair of complementary primers;
s2, carrying out double enzyme digestion on plasmid pET32a and the MYS-1 gene prepared in the step S1 by using KpnI enzyme and XhoI enzyme respectively, and then carrying out double enzyme digestion on the MYS-1 gene subjected to double enzyme digestion and the plasmid pET32a subjected to double enzyme digestion to obtain a recombinant plasmid pET32 a-MYS-1;
s3, transforming and expressing host escherichia coli E.coli BL21(DE3) by using a recombinant plasmid pET32a-MYS-1 to obtain an expression engineering bacterium pET32a-MYS-1/BL21(DE 3);
s4, inducing expression engineering bacteria pET32a-MYS-1/BL21(DE3) to express fusion protein consisting of Trx tag protein, His tag protein, collagen oligopeptide MYS-1 and the like;
s5, purifying the expressed His-tagged fusion protein by using a nickel column: after the protein is loaded, the fusion protein with the His label is specifically combined with the nickel column, and other hybrid proteins flow out because of not being specifically combined with the nickel column; gradient elution is carried out by imidazole, and the imidazole and Ni2+ are combined with a nickel column in a competitive way so as to release fusion protein and collect eluent;
s6, carrying out enzyme digestion on the fusion protein and purifying the target polypeptide MYS-1;
s7, purifying and preparing collagen oligopeptide MYS-1 by using a high performance liquid chromatography technology to obtain high-purity gene recombination high-stability collagen oligopeptide MYS-1;
wherein the primers in step S1 are:
primer 1:
primer 2:
GGTACCis a KpnI restriction enzyme cutting site,CTCGAGis XhoI restriction site;
the conditions of the PCR reaction in step S1 are: hold 95 ℃ for 5 minutes, perform 30 temperature cycles of 1 minute 95 ℃ to 1 minute 50 ℃ and then 1 minute 70 ℃ and finally hold 70 ℃ for 10 minutes.
2. The method for preparing a genetically recombinant collagen oligopeptide according to claim 1, wherein step S5 comprises using a buffer solution and a washing buffer solution, wherein the buffer solution comprises: 50mm na2HPO4, 0.3m nacl, pH 8.0;
the wash buffer comprises the following components: 50mM Na2HPO4, 0.3M NaCl, 10-50M Mimidazoles, pH 8.0.
3. The method for preparing a recombinant collagen oligopeptide according to claim 1, wherein the composition of the solution for eluting nickel column in step S5 is preferably as follows: 50mm na2HPO4, 0.3m nacl, 250m imidazole, pH 8.0.
4. The method for preparing a recombinant collagen oligopeptide according to claim 1, wherein in step S6, the steps of enzyme digestion of the fusion protein and purification of the target polypeptide MYS-1 are as follows:
(1) dialyzing the fusion protein eluate in 1 XPBS (pH7.6) buffer solution, adding enterokinase (EK, 5 IU/. mu.L) into the dialyzed fusion protein solution, wherein 5U enterokinase is added into every 500. mu.g of fusion protein, and the enzyme digestion conditions are as follows: enzyme digestion is carried out for 6h at the temperature of 25 ℃;
(2) and (2) passing the solution system after enzyme digestion through a balanced Ni-NTA purification column, collecting penetrating liquid, eluting the label protein and other bound hybrid proteins on the column by using an elution buffer solution containing 250mmol/L of imidazole, wherein the total elution volume is 5 times of that of a column bed, regenerating the Ni-NTA purification column, and collecting the penetrating liquid which is a primary purified product containing collagen oligopeptide MYS-1.
5. The method for preparing a genetically recombinant collagen oligopeptide according to claim 1, wherein in step S7, the step of purifying the collagen oligopeptide MYS-1 by high performance liquid chromatography comprises the following steps:
A. the mobile phase A is obtained by adding trifluoroacetic acid (TFA) to 100% by volume of acetonitrile, and the final concentration of TFA is 0.1% by volume; mobile phase B trifluoroacetic acid (TFA) was added to 100% water at a final TFA concentration of 0.1% by volume; the flow rate is 1.0mL/min, and the linear gradient elution is carried out for 20 min;
B. collecting the elution peak of collagen oligopeptide MYS-1 from 55-80% (v/v) of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 218 nm; and carrying out mass spectrum identification.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109593127A (en) * | 2018-12-10 | 2019-04-09 | 暨南大学 | Genetic recombination collagen sample peptide MJLGG-34 and the preparation method and application thereof |
| CN111057144A (en) * | 2019-12-31 | 2020-04-24 | 广州暨大美塑生物科技有限公司 | Gene recombination high-stability collagen oligopeptide MYS-1 and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593127A (en) * | 2018-12-10 | 2019-04-09 | 暨南大学 | Genetic recombination collagen sample peptide MJLGG-34 and the preparation method and application thereof |
| CN111057144A (en) * | 2019-12-31 | 2020-04-24 | 广州暨大美塑生物科技有限公司 | Gene recombination high-stability collagen oligopeptide MYS-1 and preparation method and application thereof |
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