CN111793568B - Pichia pastoris and application thereof - Google Patents
Pichia pastoris and application thereof Download PDFInfo
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- CN111793568B CN111793568B CN201910281043.4A CN201910281043A CN111793568B CN 111793568 B CN111793568 B CN 111793568B CN 201910281043 A CN201910281043 A CN 201910281043A CN 111793568 B CN111793568 B CN 111793568B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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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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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention discloses pichia pastoris and application thereof. The preservation number of the Pichia pastoris is CCTCC NO: M2016763. Compared with the same strain in the prior art, the polypeptide expression quantity of the Pichia pastoris strain is greatly improved, and the improvement range can reach 36.32%. In addition, the expression product of the Pichia pastoris has obvious enhancement effect on the expression quantity of the zonulin ZO-1, the laminin and the fibronectin, and can be used for preparing the gene expression up-regulator of the zonulin ZO-1, the laminin and the fibronectin.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to pichia pastoris and application thereof.
Background
The space breeding mainly induces the genetic variation of organisms through space comprehensive environmental factors such as strong radiation, microgravity, high vacuum and the like. On one hand, space environment has various factors for inducing biological mutation, space mutation breeding is an effective breeding means, and the research and development period of new varieties can be greatly shortened. On the other hand, the spatial environment has important influence on the morphological structure, growth rate, metabolic activity, gene expression and the like of microorganisms. The microorganism has small volume, simple culture condition and easy mutation compared with higher organisms, and is a better material in space biology research.
Yeasts are a group of single-cell eukaryotic microorganisms of a huge population. At present, a yeast expression system is one of commonly used eukaryotic expression systems, has been widely used for expression of exogenous genes, has broad prospects in the aspect of protein drug development and application, and has been used for clinical treatment such as insulin-like growth factors, human serum albumin and the like. The yeast expression system has the advantages of expressing exogenous genes: the method has the characteristics of fast growth, low culture cost and simple genetic operation of prokaryotes, and has post-translational processing and modification functions of mammalian cells, such as disulfide bond formation, protein phosphorylation, glycosylation and the like, so that the activity and stability of biological products can be maintained by utilizing a yeast expression system; no toxin is produced, and the safety and reliability are realized; can secrete and express, and is favorable for purification. Pichia pastoris (Pichia pastoris) can use methanol as a carbon source, is an excellent exogenous gene expression system, has wide application in the field of biological pharmacy, and is one of the most effective exogenous protein expression systems accepted in recent years.
Although a plurality of strains are carried out in space at present, no study on space mutagenesis is carried out on an excellent genetic engineering expression system of Pichia pastoris. Whether the spatial environment can improve the expression level of exogenous genes of the pichia pastoris or not is yet to be explored, and biochemical and molecular biological mechanisms of spatial action, including whether differential expression of genes in the pichia pastoris is induced from aspects of transcription factors, regulatory proteins and the like, are not yet deeply discussed and explained.
In addition, three proteins, i.e., fibronectin, are expressed in the epidermis layer, the dermis junction and the dermis layer of the skin, and the expression level of one or more of these three proteins decreases with age, resulting in an increase in wrinkles on the skin. However, few substances capable of simultaneously enhancing the expression of these three proteins have been reported in the prior art so far.
Disclosure of Invention
The invention aims to solve the technical problem of lacking substances for improving gene expression of zonulin ZO-1, laminin and/or fibronectin in the prior art, and provides pichia pastoris and application thereof. Compared with the same strain in the prior art, the polypeptide expression quantity of the Pichia pastoris strain is greatly improved, and the improvement range can reach 36.32%. In addition, the expression product of the Pichia pastoris has obvious enhancement effect on the expression quantity of the zonulin ZO-1, the laminin (especially the laminin 332) and the fibronectin, and can be used for preparing the gene expression up-regulator of the zonulin ZO-1, the laminin and the fibronectin.
The inventor selects a strain, namely space yeast (JBA-SP 02-CK-01-Gly-01) before carrying, the patent preservation number is CGMCC NO:13476, space environment mutagenesis transformation is carried out on the strain SP02-CK-01 by using a space mutagenesis method through a practical ten-number experimental satellite platform, and a mutagenized strain SP02-DZ-08 with high oligopeptide expression level, good activity and high purity is obtained unexpectedly, namely second-generation space yeast (JBA-SP 02-DZ-08-Gly-01), and the preservation number is CCTCC NO: M2016763. The inventor has unexpectedly found that the Pichia pastoris fermentation product filtrate can improve the signal expression quantity of the zonal junction protein ZO-1, the laminin and the fibronectin, and the three proteins can comprehensively strengthen the bonding compactness from three layers of the epidermis layer, the dermis layer junction and the dermis layer of the skin, and enhance the connection bonding-communication-transportation of skin cells, so that the space yeast fermentation product filtrate has good skin care efficacy.
One of the technical schemes for solving the technical problems is as follows: pichia pastoris with the preservation number of CCTCC NO: M2016763.
The second technical scheme for solving the technical problems is as follows: a gene expression up-regulator of zonulin ZO-1, laminin and/or fibronectin, said gene expression up-regulator comprising: culturing the Pichia pastoris in a culture medium, obtaining a fermentation product, centrifuging and collecting a supernatant. The laminin is preferably laminin 332.
The culture medium may be a conventional culture medium for culturing pichia pastoris, such as YPD, PDB, MEB, BMMY, BMGY, SOC and LB.
Preferably, the culture medium is BMMY culture medium; the BMMY medium comprises 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer, 1.34wt% YNB, 4×10 -5 wt% biotin (vitamin H) and 0.5v/v% methanol, the balance being water; the pH of the potassium phosphate buffer was 6.0.
The time of the culture may be conventional in the art, preferably 2 to 4 days.
The temperature of the culture may be conventional in the art, preferably 25 to 30 ℃.
The culture is preferably shake culture, and the speed of the shake culture is preferably 200 to 300rpm, more preferably 250rpm.
The inoculum size of the culture is preferably 2 to 10v/v%, more preferably 5v/v%.
Preferably, 0.1-2.0 v/v% methanol is added every 24 hours during the culture process; more preferably, 0.5v/v% methanol is added every 24 hours.
Preferably, the method further comprises the step of seed culture using a seed culture medium before said culturing; the time of the seed culture is preferably 16-18 hours;
the temperature of the seed culture is preferably 30 ℃.
The seed culture is preferably shake culture, and the rotation speed of the shake culture is preferably 250rpm.
The third technical scheme for solving the technical problems is as follows: use of pichia pastoris as described above for the preparation of a gene expression up-regulator for the zonulin ZO-1, laminin and/or fibronectin. The laminin is preferably laminin 332.
The application is preferably that the Pichia pastoris is cultivated to obtain a fermentation product, and the fermentation product is used for preparing the gene expression up-regulator.
Among them, the gene expression upregulation is preferably used in cosmetics or skin care products.
The fourth technical scheme for solving the technical problems is as follows: a process for preparing the up-regulator of gene expression of tight-linked protein ZO-1, laminin and/or fibronectin includes culturing Pichia pastoris to obtain its fermented product, centrifugal separation and collecting supernatant.
The laminin is preferably laminin 332.
The specific parameters of the preparation method are defined in detail in the preparation method of the gene expression upregulation.
As above, the gene expression upregulators are preferably used in cosmetics or skin care products.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
compared with the same strain in the prior art, the extracellular polypeptide expression quantity of the Pichia pastoris strain is greatly improved, and the improvement range can reach 36.32%; the growth vigor is improved and can be 1.42 times of that of the existing homologous strain. In addition, the expression product of the Pichia pastoris has obvious enhancement effect on the expression quantity of the zonulin ZO-1, the laminin and the fibronectin, and can be used for preparing the gene expression up-regulator of the zonulin ZO-1, the laminin and the fibronectin.
Biological material preservation information
The space yeast JBA-SP02-DZ-08-Gly-01 of the invention is preserved in China Center for Type Culture Collection (CCTCC) at 12-18 of 2016, and the preservation address is as follows: the university of martial arts collection, postal code, in the marchand district of the city of marchand in Hubei province: 430072, deposit number: CCTCC No. M2016763, culture name JBA-SP02-DZ-08-Gly-01, classification name Pichia pastoris.
Drawings
FIG. 1 shows the result of crystal violet staining of SP02-CK-01 strain.
FIG. 2 shows the results of crystal violet staining of SP02-DZ-08 strain.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 identification of essential characteristics of space yeast
1. Culture medium
Seed medium (BMGY medium): 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer (pH 6.0), 1.34wt% YNB (no amino yeast nitrogen source), 4X 10 -5 wt% biotin (vitamin H) and 1v/v% glycerol, the balance being water.
Induction medium (BMMY medium): 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer (pH 6.0), 1.34wt% YNB, 4X 10 -5 wt% biotin (vitamin H) and 0.5v/v% methanol, the balance being water.
2. Culture conditions
Culturing in seed culture medium at 30 deg.c and 250rpm for 16-18 hr to OD 600 Centrifuging at 4000rpm for 2min after the bacterial strain is 2-6 to obtain bacterial strain; the cells were inoculated in an inoculum size of 5% (volume fraction) into an induction medium at 30℃and 250rpm for 48 hours, and 0.5v/v% methanol was added every 24 hours.
3. Morphological feature contrast
SP02-CK-01 and SP02-DZ-08 were cultured on YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder) at 28℃for 3 to 4 days, and after taking a bacterial sample smear, staining with crystal violet (1%), the morphological characteristics of SP02-DZ-08 and SP02-CK-01 cell growth were compared with each other and photographed by an optical microscope, as shown in FIGS. 1 and 2.
The SP02-CK-01 strain is usually (5.0-6.0) mu m x (3.5-4.0) mu m, cells grown in YPD culture medium are densely grown, and the microscopic examination shows that the reproduction mode is multipotent budding reproduction, the morphology is regular, and the cells are uniformly colored and take an oval shape.
The SP02-DZ-08 strain is usually (6.0-6.5) mu m x (4.0-5.0) mu m, cells growing in YPD culture medium are densely grown, the microscopic examination shows that the reproduction mode is multiport budding reproduction, the morphology is more regular, the cells are uniformly colored, and the cells are oval or spherical.
Overall analysis, there was a certain difference in morphology and size between the two (table 1).
TABLE 1 morphological and size differences between starting bacteria and carried bacteria
| Sequence number | Strain | Morphology of the product | Size (mum) |
| 1 | SP02-CK-01 | Regular oval shape | (5.0～6.0)×(3.5～4.0) |
| 2 | SP02-DZ-08 | More regular, oval or spherical | (6.0～6.5)×(4.0～5.0) |
4. Culture characteristics
The SP02-CK-01 strain grows well on YPD plates, is round in colony, large in size, creamy in color, opaque, free of pigment generation, smooth in surface, slightly convex in the center, moist and easy to pick up.
The SP02-DZ-08 strain grows plump and plump on YPD plates, and has round colony, larger colony, milky white or creamy color, opalescence, no pigment generation, smooth surface, slightly convex center, wetting and easy picking.
5. Extracellular polypeptide expression level
And obtaining second-generation space yeast SP02-DZ-08 after the SP02-CK-01 is subjected to a space mutagenesis experiment. The expression level of extracellular polypeptide was measured by ELISA method using SP02-CK-01 as positive control, and extracellular samples of each strain were repeated in parallel 4 times. The average expression level of SP02-CK-01 was 1.282, and the average expression level of the SP02-DZ-08 polypeptide was increased by 36.32% (the culture conditions were "2" as described above).
Example 2 comparison of Activity before and after space Yeast mounting
1. The glycerol tube deposited bacteria of SP01-DZ-49, SP02-CK-01 and SP02-DZ-08 are inoculated into 10ml of liquid PDB culture medium according to the inoculation amount of 1 per mill, and are cultured for 24 hours at 28 ℃ and 200 rpm;
2. taking enough three bacterial liquids, centrifuging at 4 ℃ and 400rpm for 10min to remove supernatant, and re-suspending with sterile physiological saline to obtain bacterial suspension. The suspensions were diluted with physiological saline and subjected to various concentration gradients (total volume 200. Mu.L) in 96-well plates, and OD of each suspension was recorded 600 A dilution ratio of approximately 1.0;
3. according to the quantitative dilution volume, adding three bacterial suspensions into 2.0mL PDB in a 12-hole plate respectively, keeping the total volume unchanged by 3.0mL, and supplementing the rest with physiological saline, wherein 3 bacterial strains are parallel;
4. record OD 0h 600 Stationary culturing at 28deg.C in a constant temperature incubator for 22h, and measuring OD 600 The same starting concentration of the three strains was compared and the biomass difference in the late log phase of growth was compared under the same culture conditions.
OD 600 The difference in values indicates (Table 2) that aeroyeast growth activity before and after loading was stronger under the same growth conditions, and higher biomass could be achieved than that of SP 01-DZ-49: SP02-CK-01 and SP02-DZ-08 are 1.11 times and 1.42 times that of SP01-DZ-49 respectively, so that the growth vigor is stronger; front and rear mounted navigationThe difference exists between the space yeasts, and the 22h biomass of SP02-DZ-08 is 1.28 times that of SP02-CK-01, which indicates that the growth activity of the carried space yeasts is further improved under the environment of low gravity, strong radiation and strong cosmic magnetic field in the universe.
TABLE 2 comparison of growth vigor of different strains
The preservation number of the control microzyme SP01-DZ-49 is CCTCC NO: M2015725, and the related patent application is CN 105420132A.
EXAMPLE 3 Pichia pastoris fermentation product preparation
1. Preparing a culture medium: preparing a seed culture medium and an induction culture medium according to the growth requirement of saccharomycetes SP02-DZ-08, sterilizing at 115 ℃ for 20min under high temperature and high pressure, and cooling for later use.
2. Activating strains: picking up the strain at-80 ℃ for preservation, placing the strain into a liquid seed culture medium, culturing the strain for 16-18 hours at 30 ℃ and 250rpm in a shaking table, and activating the strain.
3. And (3) strain purification: the bacterial liquid after the activation is diluted in a gradient way and is plated, so that single bacterial colonies are obtained.
4. And (3) strain expansion culture: and (3) selecting a single colony in the upper plate, inoculating the single colony into a liquid BMGY culture medium, and culturing for 16-18 hours at the temperature of 250rpm by a shaking table to obtain a fermentation bacterium seed bacterial liquid.
5. And (3) yeast inoculation fermentation: the yeast liquid after the expansion culture [ the volume percent (V/V) of inoculation is 5% ] is added into the sterilized BMMY culture medium (volume 5L), and the culture is carried out for 48 hours at the constant temperature of 250rpm by a shaking table 30 ℃ and 0.5% V/V methanol is added every 24 hours.
6. And (3) clarifying: finely filtering the fermented product to obtain supernatant, namely the fermentation filtrate of Pichia pastoris SP02-DZ-08.
7. And (3) sterilization: and (3) performing high-temperature instant sterilization on the yeast fermentation filtrate to obtain a final product.
Example 4 in vitro cell efficacy test
1. Experimental model
Isolated skin; skin information: women/40 years old; the quantitative method comprises the following steps: and (5) performing a combined image analysis.
2. Test method
The test was performed using an ex vivo skin culture, the ex vivo skin being taken from the remaining skin of the surgical procedure. Firstly cleaning the isolated skin to remove redundant adipose tissues, and then punching by a puncher to form a circular skin patch with uniform aperture. Skin tissue culture was performed using normal medium. The inserted culture dish is placed in a cell pore plate, a culture medium is added in the cell pore plate, the skin sheet is placed in the inserted culture dish, the dermis layer is immersed in the culture medium, and the epidermis layer contacts air to form an air-liquid exchange interface. The sample prepared in example 3 was added to the isolated skin medium (concentration 0.1% by volume) twice daily and incubated for 5 days (37 ℃ C., 5% CO) 2 ). Freezing and embedding the skin, performing immunohistochemical staining of target protein, photographing, and performing quantitative analysis on images. The 3 target proteins analyzed were the zonulin ZO-1, laminin Lamin 332 and Fibronectin fibronectins, respectively. And carrying out quantitative analysis processing on the images according to the intensity and the area of the immunofluorescence signals. Comparing the fluorescent signal expression level of the sample group and the blank group (the blank group is selected from the corresponding ferment of the "control microzyme" SP01-DZ-49 in the examples 2 and 3), setting the signal expression level of the blank group (NT) as 100%, and the signal expression level of the sample group is more than 100% to have the promoting effect.
3. Experimental results
TABLE 3 comparison of target protein Signal expression levels
Compared with a blank control group, the filtrate of the space yeast fermentation product has obvious enhancement effect on the signal expression quantity of 3 target proteins, and promotes the structural compactness between skin cells and structures of three layers. The inventor reserves Pichia pastoris SP02-DZ-08 in China Center for Type Culture Collection (CCTCC) on 12 months of 2016 with the reservation number: cctccc No. M2016763.
Claims (11)
1. A pichia pastoris fermentation product, characterized in that the method of preparation comprises: culturing Pichia pastoris (CCTCC NO: M2016763) with preservation number of CCTCC NO in a culture medium, obtaining a fermentation product, centrifuging, and collecting a supernatant;
the culture medium is BMMY culture medium, which comprises 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer solution, 1.34wt% YNB, 4×10 -5 Biotin in weight percent and methanol in 0.5v/v percent, the balance being water; the pH of the potassium phosphate buffer was 6.0.
2. The pichia pastoris fermentation product of claim 1, wherein the incubation time is between 2 and 4 days;
the temperature of the culture is 25-30 ℃;
the culture is shake culture;
and/or the inoculation amount of the culture is 2-10 v/v%.
3. The pichia pastoris fermentation product of claim 2, wherein the shake cultivation is at a speed of 200 to 300rpm;
and/or, the inoculum size of the culture is 5v/v%.
4. The pichia pastoris fermentation product of claim 3, wherein the shake culture is at a speed of 250rpm.
5. The pichia pastoris fermentation product of any one of claims 1-4, wherein 0.1 to 2.0v/v% methanol is fed every 24 hours during the cultivation;
and/or, before said culturing, further comprising the step of seed culturing using a seed medium; the time of the seed culture is 16-18 hours, and the temperature of the seed culture is 30 ℃.
6. The pichia pastoris fermentation product of claim 5, wherein 0.5v/v% methanol is supplemented every 24 hours during the cultivation.
7. The pichia pastoris fermentation product of claim 5, wherein the seed culture is shake culture.
8. The pichia pastoris fermentation product of claim 7, wherein the shake culture is at a speed of 250rpm.
9. Application of Pichia pastoris in preparing gene expression upregulation of zonulin ZO-1, laminin and/or fibronectin; the method is characterized in that the preservation number of the pichia pastoris is CCTCC NO: M2016763; the laminin is laminin 332.
10. The use of claim 9, wherein said pichia pastoris is cultivated to obtain a fermentation product thereof, and said fermentation product is used to prepare said gene expression upregulating agent.
11. The use according to claim 9 or 10, wherein the gene expression up-regulator is suitable for use in cosmetics or skin care products.
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| CN102276707A (en) * | 2011-07-29 | 2011-12-14 | 华南理工大学 | Pichia pastoris wall protein Gcw19 as well as surface display system and construction method thereof |
| CN108823111A (en) * | 2018-06-15 | 2018-11-16 | 山东圣琪生物有限公司 | One plant has the active Pichia pastoris of endotoxin detoxification and its application |
| CN111763705A (en) * | 2019-04-01 | 2020-10-13 | 伽蓝（集团）股份有限公司 | Preparation method and application of ginsenoside composition |
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| CN102276707A (en) * | 2011-07-29 | 2011-12-14 | 华南理工大学 | Pichia pastoris wall protein Gcw19 as well as surface display system and construction method thereof |
| CN108823111A (en) * | 2018-06-15 | 2018-11-16 | 山东圣琪生物有限公司 | One plant has the active Pichia pastoris of endotoxin detoxification and its application |
| CN111763705A (en) * | 2019-04-01 | 2020-10-13 | 伽蓝（集团）股份有限公司 | Preparation method and application of ginsenoside composition |
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| Title |
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| Development of a general defined medium for Pichia pastoris;Catherine B Matthews et al.;《Biotechnol Bioeng》;第103-113页 * |
| 黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达;周亚凤等;《生物工程学报》;第400-405页 * |
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