CN117946251A - Preparation method and application of recombinant humanized III type collagen - Google Patents
Preparation method and application of recombinant humanized III type collagen Download PDFInfo
- Publication number
- CN117946251A CN117946251A CN202410124498.6A CN202410124498A CN117946251A CN 117946251 A CN117946251 A CN 117946251A CN 202410124498 A CN202410124498 A CN 202410124498A CN 117946251 A CN117946251 A CN 117946251A
- Authority
- CN
- China
- Prior art keywords
- collagen
- type iii
- recombinant human
- iii collagen
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 44
- 108010035532 Collagen Proteins 0.000 title claims abstract description 44
- 229920001436 collagen Polymers 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 102000001187 Collagen Type III Human genes 0.000 claims abstract description 19
- 108010069502 Collagen Type III Proteins 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 9
- 239000002537 cosmetic Substances 0.000 claims abstract description 5
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 210000003527 eukaryotic cell Anatomy 0.000 claims abstract 3
- 210000004027 cell Anatomy 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 12
- 241000235058 Komagataella pastoris Species 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000825 pharmaceutical preparation Substances 0.000 claims 2
- 229940127557 pharmaceutical product Drugs 0.000 claims 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 24
- 238000000855 fermentation Methods 0.000 abstract description 21
- 230000004151 fermentation Effects 0.000 abstract description 21
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 3
- 230000036541 health Effects 0.000 abstract description 3
- 230000012292 cell migration Effects 0.000 abstract 1
- 230000004663 cell proliferation Effects 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000013508 migration Methods 0.000 abstract 1
- 230000028327 secretion Effects 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 12
- 239000007788 liquid Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 108010084455 Zeocin Proteins 0.000 description 8
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 108010025188 Alcohol oxidase Proteins 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000005431 Molecular Chaperones Human genes 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000021472 generally recognized as safe Nutrition 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000033444 hydroxylation Effects 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- LXJXRIRHZLFYRP-UHFFFAOYSA-N glyceraldehyde 3-phosphate Chemical compound O=CC(O)COP(O)(O)=O LXJXRIRHZLFYRP-UHFFFAOYSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application provides a preparation method and application of recombinant human-derived type III collagen, and the amino acid sequence of the recombinant human-derived type III collagen is shown as SEQ ID NO. 1. Compared with natural type III collagen, the recombinant human type III collagen has a three-level spiral structure, shows stronger stability and has the property of approaching to the natural collagen. Can be expressed and prepared in higher eukaryotic cells such as yeast, has good biological activity superior to natural collagen, and can effectively promote cell proliferation and migration; can be widely applied to the fields of foods, cosmetics, health products, medical instruments and the like. The yeast fermentation method of the application does not need methanol, can be used for safe and high-density fermentation production, has extremely low culture cost, short period and high protein expression, can be used for large-scale industrial production, and can be used for extracellular secretion of collagen by an expression system.
Description
Technical Field
The invention relates to the fields of molecular biology technology and gene recombination, in particular to recombinant human type III collagen produced by pichia pastoris and application thereof.
Background
Collagen (collagen) is the most abundant protein in the human body, the main component of extracellular matrix (ECM), and the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Its main functions are maintaining extracellular environment, maintaining normal physiological functions of tissue and organ and repairing injury of body. Collagen is a natural biological resource, has excellent biocompatibility which is incomparable with other high molecular materials, has supporting elasticity and degradability for cells, has important functions for maintaining normal physiological functions and damage repair of cells, tissues and organs, and is widely applied to the industries of medicines, health care products and cosmetics.
Collagen products sold in the current market are all taken from animal tissues such as pigs, cows, fishes and the like, are difficult to avoid virus infection, can cause immunological rejection and allergic symptoms, are not soluble in water, are nonuniform in property, are difficult to be utilized by human bodies, and can be used after being treated by chemical means. Can lead to immunological rejection and allergic symptoms. Therefore, the collagen can only be used in cosmetics and health care products, and the original biological functions of the collagen cannot be exerted. Along with the development of biotechnology, the recombinant collagen is obtained by a microbial fermentation method by utilizing a gene recombination technology, so that the virus hidden trouble existing in the traditional extraction method is solved, and meanwhile, compared with the natural collagen, the hydrophilicity and biocompatibility of the artificially designed recombinant collagen are obviously improved.
However, research shows that the collagen expressed by using recombinant strain and escherichia coli cannot be subjected to post-translational modification of protein, and for some recombinant human collagen with higher conformation, the collagen cannot be glycosylated after being expressed by using escherichia coli. Meanwhile, since E.coli is a prokaryote, there is no Endoplasmic Reticulum (ER), and thus there is no chaperone protein that can help collagen fold into a higher conformation and some enzymes that modify synthetic proteins such as hydroxylation, disulfide bond formation. Thus, it is difficult for the E.coli system to express triple helix collagen having a natural higher structure.
The human recombinant collagen produced by yeast fermentation overcomes the defects of animal sources and collagen expressed by escherichia coli. The yeast expression system has molecular chaperones and enzymes for post-translational modification of proteins (such as glycosylation, hydroxylation, acetylase and the like), and is very suitable for forming triple-helical collagen with a higher structure. The culture condition of the yeast cells is lower than that of the mammalian cells, and the yeast cells are very suitable for large-scale industrialized fermentation production. The yeast system is used for substituting animal-derived collagen and escherichia coli prokaryotic expression system-derived recombinant collagen, and has important research value in biomedical application.
With the use of a series of pichia expression kits developed by Invitrogen, thousands of biologically functional exogenous proteins or protein structures from bacteria, fungi, protozoa, plants, invertebrates, vertebrates including humans, viruses, etc. have been successfully expressed with this system. In recent years, pichia pastoris is recognized by the U.S. FDA as GENERALLY RECOGNIZED AS SAFE (GRAS) microorganism, so that pichia pastoris has great application potential in the food field. The expression plasmids commonly used at present for pichia pastoris are divided into pPIC series methanol induction type expression vectors (such as pPIC3.5k, pPIC9K, pPICZ, pAO815 and the like) and pGAP series constitutive expression vectors according to different promoters. Wherein the pPIC series plasmid contains Alcohol Oxidase (AOX) promoter, which is a strong promoter taking methanol as the only inducer and strictly controlled by the methanol, and the strong inducible promoter AOX can very effectively control the expression of exogenous genes because the AOX expression is regulated by the transcription level. However, the pichia pastoris has the defect that cells grow to high density under uncontrolled conditions, and then the expression of the exogenous protein is started through methanol induction, so that the culture time and the workload of engineering bacteria are increased. Meanwhile, in the large-scale production process, the use of methanol has a plurality of defects such as easy volatilization, potential safety hazard, toxicity, inapplicability to the production of medicines and food proteins and the like, thereby restricting the large-scale application of the pichia pastoris expression system. pGAP series plasmid contains 3-phosphoglyceraldehyde dehydrogenase (GAP) promoter, the GAP promoter is regulated and controlled by carbon source, the constitutive promoter does not need methanol induction, the fermentation process is simple, and the method has the advantage of being suitable for food scale application. However, compared with the GAP promoter of the AOX promoter, the level of the expressed foreign protein is lower, the GAP plasmid is transformed into a high-copy free expression vector by adding the yeast autonomous ARS element, so that the recombinant human III type collagen can be safely and non-toxic produced, and the yield of the recombinant human III type collagen can be greatly improved.
Disclosure of Invention
In view of the above, the present invention provides a recombinant human type III collagen and its application. The recombinant protein has a three-level spiral structure, shows stronger stability and has the property of approaching to natural collagen.
A safe non-methanol episomal autonomous replication high-copy pGAPZalpha A-ARS vector of Pichia pastoris, which can safely produce target recombinant protein with high yield by transforming GAP plasmid into a high-copy episomal expression vector.
A recombinant human-derived III type collagen is characterized by having an amino acid sequence shown as SEQ ID NO. 1; or has amino acid sequence with the same protein function by substituting, deleting or adding amino acid in the amino acid sequence shown in SEQ ID NO. 1.
In some embodiments, the substituted, deleted, or added amino acid is one or more.
The invention also provides a nucleic acid for encoding the recombinant human III type collagen.
In some embodiments, the nucleotide sequence of the nucleic acid encoding the recombinant human type III collagen shown in SEQ ID NO.1 is shown in SEQ ID NO. 2.
The invention also provides a recombinant vector comprising the nucleic acid of the invention.
In the invention, the recombinant vector also comprises a skeleton vector; wherein, the framework vector is preferably pGAPZalpha A.
The invention also provides a host transformed with the recombinant vector.
The invention also provides application of the recombinant human-derived III-type collagen, the nucleic acid of claim 5 or 6, the recombinant vector or the host in preparation of anti-aging and moisturizing products.
The invention also provides a product comprising the recombinant human III type collagen.
In the invention, the product is in the form of powder solid, and can be processed into powder injection, granules, oral liquid, injection or capsules later.
The recombinant humanized III type collagen provided by the invention has an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence in which an amino acid is substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.1 without changing the activity of the protein. The collagen has a three-level spiral structure, shows good stability, has the property of being similar to natural collagen, can be effectively and stably expressed in a large amount in pichia pastoris, is easy to obtain a high-purity product, and can be suitable for industrialized mass production.
Table 1, SEQ ID NO.1 amino acid sequence, SEQ ID NO.2 nucleotide sequence:
Detailed Description
The invention provides a recombinant human III type collagen produced by a high-copy free expression vector and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Examples
The experimental method comprises the following steps:
(1) Construction of recombinant vector pGAPZαA-ARS:
The ARS sequence synthesized by chemical synthesis is utilized, ecoRI and BamHI double enzyme cutting sites are introduced and inserted into an expression vector pGAPZalpha A, so as to obtain recombinant plasmids, the recombinant plasmids are linearized and respectively transformed into escherichia coli electrically, and pGAPZalpha A-ARS plasmids are identified and extracted through colony PCR.
(2) The target gene shown in SEQ ID No.2 is utilized, hindIII and XbaI double enzyme cutting sites are introduced, the target gene is inserted into recombinant pGAPZalpha A-ARS to obtain a humanized III type collagen recombinant plasmid, the recombinant plasmid is linearized and respectively electrically transformed into Pichia pastoris KM71, and after colony PCR identification and sequencing identification, glycerol and industrial glucose are used for induced expression.
(3) Shake flask fermentation expression of recombinant human-derived type III collagen:
BMGY medium was used: 5.0g/L yeast powder, 15.0g/L soybean peptone, 13.4g/L YNB, 15.0g/L glycerol glucose, and culturing at 28deg.C and 250rpm for 96 hr, and adding glycerol every 36 hr. And (5) centrifuging to collect thalli and fermentation supernatant respectively.
(4) Recombinant yeast cells:
The fermentation broth was centrifuged at 12000rpm for 10min to collect the cells, washed three times with pure water, transferred into a 50ml centrifuge tube, added with a proper amount of lysate (20 mM Tris-HCl, 50mmol/L KH2PO4,1mmol/L EDTA-2Na, 5% glycerol by volume, pH 7.4), after mixing uniformly, sonicated, after disruption was completed, the lysate was centrifuged at 12000rpm/min for 15min, and then analyzed by SDS-PAGE.
(5) Fermentation in upper tank of fermentation tank
And (3) absorbing 500 mu L of bacterial liquid, inoculating the bacterial liquid into 200mL of seed shake flask culture medium, culturing for 24 hours at the temperature of 30 ℃ under the condition of 200r/min, and inoculating 100mL of seeds into a 5L fermentation tank for fermentation. The fermentation initial culture medium is BMGY culture medium, and the fermentation tank is divided into a glycerol growth stage, a glycerol feed culture and a different carbon source feed culture stage. In the glycerol growth stage, DO is controlled at 35%, the temperature is controlled at 28 ℃, fed-ammonia water is controlled to control fermentation liquor to be at about pH5.5, when DO value is rapidly increased about 24 hours of culture, the fermentation liquor enters a glycerol feed culture stage, at the moment, 50% of glycerol (containing histidine 5 g/L) feed is fed, when yeast grows to OD600 of about 100, the glycerol feed is stopped, when DO value is rapidly increased again, fed-fed culture in the third stage is respectively carried out according to different carbon sources, the carbon sources respectively comprise glycerol, glucose, sucrose and mixed carbon sources (glycerol: sucrose is 1:2,1:1, 2:1), and all the carbon sources are prepared into 50% concentration (containing histidine 5.0 g/L) for fed-feeding. The various media formulations used were as described in the pichia pastoris operating manual of Invitrogen.
Analysis of results:
(1) Recombinant bacterium PCR verification result
The specific experimental operation process is as follows:
① Selecting a yeast transformant to be detected, and prefabricating a PCR template according to the following method;
② Single colonies were picked and placed in a 1.5mlEP tube containing 200. Mu.L of deionized water and heated at 100deg.C for 5min;
③ Sucking 5uL of liquid as a template, adding the template into a PCR reaction system, and performing fluorescent PCR amplification;
④ The PCR reaction system is as follows:
primer COL-F1. Mu.L
Primer COL-R1. Mu.L
2x SYBR Green：15μL
And (3) a template: 5 mu L
Make up water to total volume: 30 mu L
The PCR reaction procedure is shown in Table 1 below:
(2) High copy recombinant strain selection
PGAPZA-ARS-ReCOL in KM71 yeast chromosome is an episomal vector, and the more the copy number of pGAPZA-ARS-ReCOL is, more recombinant human type III collagen can be expressed, the higher the resistance phenotype is, and the better the growth on a Zeocin plate with higher concentration is realized. For this reason, zeocin has achieved good results in screening high copy recombinants. Finally, few strains growing on YPD plates of 10mg/ml Zeocin are obtained, namely high-copy recombinant strains.
The specific operation is as follows:
① Selecting hundreds of single colonies which grow well on YPD medium plates, and photocopying the single colonies on YPD medium plates containing different concentrations of Zeocin, so as to ensure that YPD plates containing higher concentrations of Zeocin are photocopied first and then YPD plates containing lower concentrations of Zeocin are photocopied;
② The photocopied flat plate is placed in an incubator at 30 ℃ in an inverted mode for 2-5 d;
③ For transformants on YPD plates without Zeocin, larger colonies were grown at 1 d; whereas for transformants on YPD plates containing different concentrations of Zeocin, it was possible to observe a distinct single colony after 4d and even after 6 d.
(3) Fermentation supernatant and in-cell protein analysis
The specific experimental operation is as follows:
① Sample treatment: adding 5X Loadingbuffer mu l of the mixture respectively, mixing uniformly, boiling for 5-10 min at 100 ℃ on a metal bath device to denature protein, centrifuging for 10min at 10,000rpm/min, and taking the supernatant for electrophoresis;
② After the preparation of the separation gel and the concentration gel, 20 μl of the treated sample was sampled. Pouring electrophoresis buffer solution after sample addition, and immediately performing electrophoresis (constant pressure 80V, 120V after entering separation gel);
③ After electrophoresis, taking down the gel, and transferring the film;
④ After membrane transfer, 5% skimmed milk is used for sealing at room temperature for 1-2 hours, the primary antibody is incubated, TBST is washed three times, the secondary antibody is incubated, and TBST is washed 3 times;
⑤ And mixing the luminescent liquid A and the luminescent liquid B according to a ratio of 1:1, adding the luminescent liquid A and the luminescent liquid B to the surface of the PVDF film, and shooting by using a chemiluminescent instrument.
The results show that the recombinant collagen dimer exists in the fermentation liquor, the monomer is easy to degrade, the secreted recombinant collagen has residues in the bacterial body, and meanwhile, the collagen monomer in the bacterial body is relatively stable and is not easy to degrade.
(4) Analysis of biological Activity of fermented collagen
The specific experimental operation is as follows:
① Taking NIH/3T3 cells in a logarithmic growth phase with good growth state, and inoculating 2500 cells in each hole into a 96-well plate; each group is 100 mu l and is provided with 3 compound holes, and the compound holes are placed in a cell incubator for culture;
② After 24 hours, adding collagen fermentation broth according to the groups in Table 1;
③ After culturing for 48 hours, observing the proliferation condition of the cells, and taking a picture by a microscope;
④ The culture medium containing the test substance is discarded, and a freshly prepared culture medium containing 10 mu l of cell activity detection liquid CCK8 is added into each well; ⑤ After the culture is continued for 4 hours in an incubator, detecting the absorbance OD value of each Kong Bochang nm by using an enzyme-labeled instrument; ⑥ Data analysis: taking the average value of the experimental results as a final experimental result, taking NC as a control, and carrying out statistical analysis to test by a cell experiment, wherein the fermentation broth recombinant collagen has good biological activity of promoting cell wall adhesion proliferation.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the present invention, which is also considered to be within the scope of the invention.
Claims (11)
1. The recombinant human-derived type III collagen is characterized in that the amino acid sequence of the recombinant human-derived type III collagen is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the recombinant human type III collagen of claim 1.
3. The nucleic acid molecule of claim 2, wherein the sequence of the nucleic acid molecule is set forth in SEQ ID No. 2.
4. An episomal high expression pGAPZ A-ARS vector, wherein the vector comprises the nucleic acid molecule of claim 2.
5. A host cell comprising the collagen of claim 1 or the collagen of claim
The nucleic acid molecule according to claim 2 or the vector according to claim 4.
6. The host cell of claim 5, wherein the host cell is a eukaryotic cell.
7. The host cell of claim 6, wherein the eukaryotic cell is pichia pastoris.
8. A method of preparing the recombinant human type III collagen of claim 1, comprising the steps of: expressing the recombinant human type III collagen by the host cell according to any one of claims 5 to 7, and then isolating and purifying.
9. Use of recombinant human type III collagen according to claim 1, or recombinant human type III collagen encoded by a nucleic acid molecule according to claim 2 or 3, or produced by a host cell according to any one of claims 5-7, in the preparation of a food, cosmetic or pharmaceutical product.
10. A composition comprising the recombinant human type III collagen according to claim 1, or the recombinant human type III collagen encoded by the nucleic acid molecule of claim 2 or 3, or the recombinant human type III collagen produced by the host cell of any one of claims 5-7.
11. Use of a composition according to claim 10 for the preparation of a food, cosmetic or pharmaceutical product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410124498.6A CN117946251A (en) | 2024-01-30 | 2024-01-30 | Preparation method and application of recombinant humanized III type collagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410124498.6A CN117946251A (en) | 2024-01-30 | 2024-01-30 | Preparation method and application of recombinant humanized III type collagen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117946251A true CN117946251A (en) | 2024-04-30 |
Family
ID=90791925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410124498.6A Pending CN117946251A (en) | 2024-01-30 | 2024-01-30 | Preparation method and application of recombinant humanized III type collagen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117946251A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118561987A (en) * | 2024-08-01 | 2024-08-30 | 英特菲尔(成都)生物制品有限责任公司 | Self-assembled recombinant collagen and preparation method and application thereof |
-
2024
- 2024-01-30 CN CN202410124498.6A patent/CN117946251A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118561987A (en) * | 2024-08-01 | 2024-08-30 | 英特菲尔(成都)生物制品有限责任公司 | Self-assembled recombinant collagen and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112552393B (en) | Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof | |
| EP0156355B1 (en) | Method of improving the yield of heterologous protein produced by cultivating recombinant bacteria | |
| CN105358694B (en) | Yeast promoter from pichia pastoris yeast | |
| WO2024002149A1 (en) | Recombinant type-iii collagen and method for preparing same | |
| CN117946251A (en) | Preparation method and application of recombinant humanized III type collagen | |
| CN116375847A (en) | Yeast recombinant XVII type humanized collagen and preparation method thereof | |
| CN101974587A (en) | Efficient expression method of human serum albumin | |
| CN100357441C (en) | Yeast expressing system of recombined human nerve growth factor and process for preparing recombined human nerve grouth factor | |
| CN113249241B (en) | Construction and application of saccharomyces cerevisiae protease deletion strain | |
| CN101450961B (en) | Artificial synthetic porcine interferon and method for preparing porcine interferon | |
| CN1205337C (en) | Method for culturing microorganisms having methanol metabolic pathway | |
| US20210309982A1 (en) | Materials and methods for creating strains of saccharomyces cerevisiae that exhibit an increased ability to ferment oligosaccharides into ethanol | |
| CN116509764A (en) | Yeast fermentation product filtrate containing recombinant type 17 human collagen | |
| CN107778365A (en) | The recombinant-protein expression that a kind of multiple spot is integrated | |
| CN108841852A (en) | A kind of high yield 5-ALA produces construction method and the application of bacterial strain | |
| US11913002B2 (en) | Modified plant endosperm specific promoter and use thereof | |
| CN114107176A (en) | CHO cell line for stably expressing African swine fever CD2v protein and construction method and application thereof | |
| CN118006716B (en) | Method for preparing recombinant human albumin with high expression and low O-glycosylation level | |
| CN101492674A (en) | Method for producing human amyloid A<beta>1-42 with methylotrophy yeast | |
| CN113122461A (en) | Single cell protein producing strain and its application | |
| RU2290434C1 (en) | Pichia pastoris 2-2 yeast strain as producer of human platelet growth factor (pdgf-bb) and method for production of human platelet growth factor | |
| CN114908109B (en) | Construction of acid protease expression strain suitable for feed and batch fermentation process | |
| WO2001032895A1 (en) | Recombinant yeast strain for expressing human lactoferrin, n-lobe lactoferrin, c-lobe lactoferrin and process for preparation recombinant human lactoferrin, n-lobe lactoferrin, c-lobe lactoferrin using the same | |
| CN117603365A (en) | Preparation and application of recombinant collagen I type, recombinant collagen III type and recombinant collagen VI type | |
| CN118772263A (en) | Recombinant human III type collagen capable of promoting cell migration and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |