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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- 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
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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.
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