CN111793568A

CN111793568A - Pichia pastoris and application thereof

Info

Publication number: CN111793568A
Application number: CN201910281043.4A
Authority: CN
Inventors: 李俊; 李慧; 章漳; 蒋丹丹
Original assignee: Jialan Group Co ltd
Current assignee: Jialan Group Co ltd
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2020-10-20
Anticipated expiration: 2039-04-09
Also published as: CN111793568B

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 by 36.32 percent. In addition, the expression product of the pichia pastoris has obvious enhancement effect on the expression quantity of the zon-1, laminin and fibronectin, and can be used for preparing the gene expression up-regulator of the zon-1, laminin and fibronectin.

Description

Pichia pastoris and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to pichia pastoris and application thereof.

Background

Space breeding mainly induces gene variation of organisms through space comprehensive environmental factors such as strong radiation, microgravity, high vacuum and the like. On one hand, the space environment has a plurality of factors for inducing biological mutation, and space mutation breeding is an effective breeding means, so that the research and development period of new varieties can be greatly shortened. On the other hand, the spatial environment has an important influence on the morphological structure, growth rate, metabolic activity, gene expression, etc. of the microorganism. The microorganism has small volume and simple culture condition, is easier to cause variation compared with higher organisms, and is a better material in space biological research.

Yeasts are a large group of unicellular eukaryotic microorganisms. At present, a yeast expression system is one of commonly used eukaryotic expression systems, has been widely used for the expression of exogenous genes, has a wide prospect in the development and application of protein drugs, 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 yeast cell culture medium has the characteristics of fast growth of prokaryotes, low culture cost and simple genetic operation, and also has post-translational processing and modification functions of mammalian cells, such as formation of disulfide bonds, protein phosphorylation, glycosylation and the like, so that the yeast expression system is favorable for maintaining the activity and stability of biological products; does not generate toxin, and is safe and reliable; can secrete expression, and is favorable for purification. Pichia pastoris (Pichia pastoris) can utilize methanol as a carbon source, is an excellent exogenous gene expression system, has wide application in the field of biopharmaceuticals, and is one of the most effective exogenous protein expression systems recognized in recent years.

Although a plurality of strains are carried out spatially, spatial mutagenesis is not researched on a superior genetic engineering expression system of pichia pastoris. Whether the space environment can improve the expression level of the exogenous gene of the pichia pastoris or not is yet explored, and the biochemical and molecular biological mechanisms of the space action, including whether the differential expression of the gene in the pichia pastoris is induced from the aspects of transcription factors, regulatory proteins and the like, are not further discussed and explained.

In addition, three proteins, tight junction protein, laminin and fibronectin, are expressed in the epidermis layer, the dermis layer and the dermis layer of the skin, and the expression level of one or more of the three proteins is reduced with age, thereby causing the increase of wrinkles on the skin. However, few substances capable of simultaneously enhancing the expression of the three proteins have been reported in the prior art so far.

Disclosure of Invention

The invention aims to solve the technical problem that substances for improving the gene expression of zon-1, laminin and/or fibronectin are lacked 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 by 36.32 percent. 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 an initial strain, namely, a pre-loaded space yeast (JBA-SP02-CK-01-Gly-01), the patent preservation number of which is CGMCC NO:13476, and carries out space environment mutagenesis transformation on the initial strain SP02-CK-01 by a 'practice ten' experimental satellite platform and utilizing a space mutagenesis means, thereby unexpectedly obtaining a mutagenic strain SP02-DZ-08, namely, a second-generation space yeast (JBA-SP02-DZ-08-Gly-01), the preservation number of which is CCTCC NO: M2016763, with high oligopeptide expression level, good activity and high purity. The inventor unexpectedly finds that the pichia pastoris fermentation product filtrate can improve the signal expression quantity of zon-1, laminin and fibronectin, and the three proteins can comprehensively enhance the combination tightness from the epidermal layer, the real epidermal junction and the dermal layer of the skin and enhance the connection combination-communication-transportation of skin cells, so that the space yeast fermentation product filtrate has good skin care effect.

One of the technical solutions for solving the above technical problems of the present invention is: a Pichia pastoris (Pichia pastoris) with a preservation number of CCTCC NO: M2016763.

The second technical solution of the present invention for solving the above technical problems is: a gene expression up-regulator of Claudin ZO-1, laminin and/or fibronectin, which is prepared by a method comprising: culturing the Pichia pastoris in a culture medium, obtaining a fermentation product, centrifuging and collecting a supernatant to obtain the Pichia pastoris. The laminin is preferably laminin 332.

The culture medium may be a culture medium for Pichia pastoris, which is conventional in the art, such as YPD, PDB, MEB, BMMY, BMGY, SOC, and LB medium.

Preferably, the culture medium is BMMY culture medium; the BMMY culture medium comprises 2 wt% of peptone, 1 wt% of yeast extract, 100mM potassium phosphate buffer, 1.34 wt% of YNB, and 4 x 10^-5wt% biotin (vitamin H) and 0.5 v/v% methanol, the balance being water; the pH of the potassium phosphate buffer was 6.0.

The culture time may be conventional in the art, and is preferably 2 to 4 days.

The temperature of the culture may be conventional in the art, and is preferably 25 to 30 ℃.

The culture is preferably shake culture, and the speed of the shake culture is preferably 200-300 rpm, and more preferably 250 rpm.

The amount of the inoculum for the culture is preferably 2 to 10 v/v%, more preferably 5 v/v%.

Preferably, in the culture process, 0.1-2.0 v/v% methanol is supplemented every 24 hours; more preferably, 0.5 v/v% methanol is added every 24 hours.

Before the cultivation, preferably further comprising the step of seed cultivation using a seed culture medium; the time for 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 250 rpm.

The third technical scheme for solving the technical problems is as follows: the application of the Pichia pastoris in preparing the up-regulator of the gene expression of the zonulin ZO-1, the laminin and/or the fibronectin. The laminin is preferably laminin 332.

The application is preferably to culture the pichia pastoris to obtain a fermentation product thereof, and to prepare the gene expression up-regulator by using the fermentation product.

Wherein, the gene expression up-regulator is preferably suitable for cosmetics or skin care products.

The fourth technical scheme for solving the technical problems is as follows: a preparation method of a gene expression up-regulator of zonulin ZO-1, laminin and/or fibronectin comprises the steps of culturing the Pichia pastoris, obtaining a fermentation product, centrifuging and collecting a supernatant.

The laminin is preferably laminin 332.

The specific parameters in the preparation method are defined in detail in the preparation method of the gene expression up-regulator.

As above, the gene expression up-regulator is preferably used in cosmetics or skin care products.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

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 amplitude can reach 36.32 percent; the growth vigor is improved and can be 1.42 times of that of the existing strain of the same species. In addition, the expression product of the pichia pastoris has obvious enhancement effect on the expression quantity of the zon-1, laminin and fibronectin, and can be used for preparing the gene expression up-regulator of the zon-1, laminin and fibronectin.

Biological material preservation information

The space yeast JBA-SP02-DZ-08-Gly-01 is preserved in China Center for Type Culture Collection (CCTCC) at 18 months 12 in 2016, and the preservation address is as follows: wuhan university collection center in Wuchang district, Wuhan city, Hubei province, zip code: 430072, with the preservation number: m2016763, culture name JBA-SP02-DZ-08-Gly-01, and classification name Pichia pastoris (Pichia pastoris).

Drawings

FIG. 1 shows the results of crystal violet staining microscopy of SP02-CK-01 strain.

FIG. 2 shows the results of crystal violet staining microscopy of SP02-DZ-08 strain.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

EXAMPLE 1 identification of basic characteristics of Spaceflight Yeast

1. Culture medium

Seed medium (BMGY medium): 2 wt% peptone, 1 wt% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34 wt% YNB (without amino yeast nitrogen source), 4X 10^-5wt% biotin (vitamin H) and 1 v/v% glycerol, the balance being water.

Induction medium (BMMY medium): 2 wt% peptone, 1 wt% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34% wt YNB, 4X 10^-5wt% biotin (vitamin H) and 0.5 v/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 OD₆₀₀After 2-6, centrifuging at 4000rpm for 2min to obtain thalli; the cells were inoculated in an induction medium at an inoculum size of 5% (volume fraction) and cultured at 30 ℃ and 250rpm for 48 hours, and 0.5 v/v% methanol was supplemented every 24 hours.

3. Morphological feature comparison

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 3-4 days, and then bacterial smears were stained with crystal violet (1%), and then observed by an optical microscope to compare growth morphological characteristics of SP02-DZ-08 and SP02-CK-01 cells, and photographed as shown in FIG. 1 and FIG. 2.

The SP02-CK-01 strain is usually (5.0-6.0) mum x (3.5-4.0) mum, the cells growing in the YPD medium grow densely, the reproduction mode is multi-end budding reproduction under microscopic examination, the shape is regular, the cells are uniformly colored and are oval.

The SP02-DZ-08 strain is usually (6.0-6.5) mum x (4.0-5.0) mum, the cells growing in the YPD culture medium grow densely, the reproduction mode is multi-end budding reproduction through microscopic examination, the shape is regular, the cells are uniformly colored, and the cells are in an oval shape or a spherical shape.

The two types of samples were different in morphology and size in the whole analysis (Table 1).

Table 1 shows the difference in morphology and size between the growing bacteria and the carrying bacteria

Serial number	Bacterial strains	Form of the composition	Size (mum)
				1	SP02-CK-01	Regular, oval shape	(5.0～6.0)×(3.5～4.0)
2	SP02-DZ-08	More regular, ovoid or spherical	(6.0～6.5)×(4.0～5.0)

4. Characteristics of cultivation

The SP02-CK-01 strain grows well on the YPD plate, the colony is round and large, is creamy, opaque, free of pigment, smooth in surface, slightly convex in center, moist and easy to pick up.

The SP02-DZ-08 strain grows well on the YPD plate, and the colony is round, large, milky white or creamy, opaque, free of pigment, smooth in surface, slightly convex in center, moist and easy to pick up.

5. Extracellular polypeptide expression level

SP02-CK-01 is subjected to a space mutagenesis experiment of practice No. ten to obtain second-generation space yeast SP 02-DZ-08. SP02-CK-01 is used as a positive control, the extracellular polypeptide expression quantity is determined by an ELISA method, and each strain extracellular expression sample is subjected to 4 parallel repetitions. The average expression level of SP02-CK-01 is 1.282, and the expression level of SP02-DZ-08 polypeptide is improved by 36.32% (see the culture conditions in the section "2" above).

Example 2 comparison of Activity before and after Sporobolomyces Loading

1. Inoculating glycerol tube preserved thallus of SP01-DZ-49, SP02-CK-01 and SP02-DZ-08 into 10ml of liquid PDB culture medium according to the inoculation amount of 1 per thousand, and culturing at 28 ℃ and 200rpm for 24 h;

2. taking enough three bacterial liquids, centrifuging at 4 ℃, 400rpm for 10min to remove supernatant, and resuspending with sterile normal saline to obtain bacterial suspension. The OD of each bacterial suspension was recorded by diluting with physiological saline and performing different concentration gradients (total volume 200. mu.L) for the three bacterial suspensions in a 96-well plate₆₀₀The dilution ratio is approximately 1.0;

3. adding three bacterial suspensions into a 12-well plate to 2.0mL PDB according to the above quantitative dilution volume, keeping the total volume to be 3.0mL, and filling the rest with normal saline, wherein 3 strains are parallel;

4. OD recorded 0h₆₀₀Static culture at 28 ℃ for 22h in a constant temperature incubator, and OD measurement₆₀₀And comparing the biomass difference of the three strains at the same starting concentration and the late growth log stage under the same culture condition.

OD₆₀₀The difference in the values indicates (table 2) that the aviation yeast before and after loading has stronger growth activity under the same growth conditions, and can achieve higher biomass than SP 01-DZ-49: SP02-CK-01 and SP02-DZ-08 are respectively 1.11 times and 1.42 times of SP01-DZ-49, so that the growth vigor is stronger; the difference also exists between the space yeast before and after the carrying, the biomass of SP02-DZ-08 in 22h is 1.28 times of that of SP02-CK-01, which shows that the growth vigor of the space yeast after the carrying is further improved under the environment of low gravity, strong radiation and strong magnetic field of the universe.

TABLE 2 comparison of growth vigor of different strains

The preservation number of the control yeast 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: respectively preparing a seed culture medium and an induction culture medium according to the growth requirement of the yeast SP02-DZ-08, sterilizing at 115 ℃ for 20min at high temperature and high pressure, and cooling for later use.

2. Activating strains: selecting the strain to be preserved at the temperature of minus 80 ℃, putting the strain into a liquid seed culture medium, and culturing the strain in a shaking table at the temperature of 30 ℃ and the rpm of 250 for 16 to 18 hours to activate the strain.

3. And (3) strain purification: and (4) diluting and plating the activated bacteria liquid in a gradient manner to obtain a single colony.

4. And (3) strain amplification culture: and (3) selecting a single bacterial colony in the flat plate in the previous step, inoculating the single bacterial colony into a liquid BMGY culture medium, and culturing for 16-18 hours at 30 ℃ and 250rpm of a shaking table to obtain a zymocyte seed bacterial liquid.

5. Yeast inoculation and fermentation: adding the yeast liquid after the enlarged culture (the volume percentage (V/V) of inoculation is 5 percent) into a sterilized BMMY culture medium (the volume is 5L), culturing for 48 hours at the constant temperature of 250rpm by a shaking table at 30 ℃, and supplementing 0.5 percent of V/V methanol every 24 hours.

6. Clarification treatment: and finely filtering the fermented product to obtain supernatant, namely the pichia pastoris SP02-DZ-08 fermentation filtrate.

7. And (3) sterilization: and (4) instantly sterilizing the yeast fermentation filtrate at high temperature to obtain a final product.

Example 4 in vitro cell efficacy assay

1. Experimental model

Skin ex vivo; skin information: female/40 years of age; the quantitative method comprises the following steps: and (4) organized image analysis.

2. Test method

The test was performed using ex vivo skin cultures, which were taken from the skin remaining from the surgical procedure. The in vitro skin is cleaned to remove redundant adipose tissues, and then a puncher is used for punching to form a round skin sheet with uniform aperture. The skin tissue culture is carried out by adopting a normal culture medium. Putting the insertion type culture dish into a cell pore plate, adding a culture medium into the cell pore plate, putting a skin sheet into the insertion type culture dish, immersing the dermis layer in the culture medium, and contacting the epidermis layer with air to form an air-liquid exchange interface. The sample prepared in example 3 was added twice daily to the in vitro skin culture medium (concentration 0.1%, volume percent) and cultured for 5 days (37 ℃, 5% CO)₂). And (3) carrying out freezing embedding treatment on the skin, carrying out immunohistochemical staining on the target protein, and carrying out quantitative analysis on the image after photographing. The 3 target proteins analyzed were zonulin ZO-1, Laminin lamin 332, and Fibronectin. And carrying out image quantitative analysis processing according to the intensity and the area of the immunofluorescence signal. The fluorescence signal expression levels of the sample group and the blank control group (here, the blank control group, selected from the corresponding fermentation products of "control yeast" SP01-DZ-49 in examples 2 and 3) were compared, and the signal expression level of the blank control group (NT) was set to 100%, and the signal expression level of the sample group greater than 100% was considered to have an accelerating effect.

3. Results of the experiment

TABLE 3 comparison of target protein signal expression

Compared with a blank control group, the space yeast fermentation product filtrate 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 deposits Pichia pastoris SP02-DZ-08 in the China Center for Type Culture Collection (CCTCC) at 2016, 12 months and 18 days, with the deposit numbers as follows: CCTCC NO: M2016763.

Claims

1. Pichia pastoris (Pichia pastoris) with a preservation number of CCTCC NO: M2016763.

2. A method for preparing a gene expression up-regulator of Claudin ZO-1, laminin and/or fibronectin, comprising: culturing the Pichia pastoris of claim 1 in a culture medium, obtaining a fermentation product, centrifuging and collecting the supernatant;

the laminin is preferably laminin 332.

3. The gene expression up-regulator of claim 2, wherein the culture medium is BMMY culture medium comprising 2 wt% peptone, 1 wt% yeast extract, 100mM potassium phosphate buffer, 1.34 wt% YNB, 4X 10^-5wt% biotin and 0.5 v/v% methanol, the balance being water; the pH of the potassium phosphate buffer is 6.0;

the culture time is 2-4 days;

the culture temperature is 25-30 ℃;

the culture is shake culture, wherein the speed of the shake culture is preferably 200-300 rpm, and more preferably 250 rpm;

and/or the inoculation amount of the culture is 2-10 v/v%, preferably 5 v/v%.

4. The gene expression up-regulator according to claim 2 or 3, wherein 0.1 to 2.0 v/v% methanol is supplemented every 24 hours during the culture; preferably, 0.5 v/v% methanol is supplemented every 24 hours;

and/or, before the culture, further comprising the step of seed culture by using a seed culture medium; the time of seed culture is 16-18 hours, and the temperature of seed culture is 30 ℃.

5. The gene expression up-regulator according to claim 4, wherein the seed culture is shake culture, and the rotation speed of the shake culture is preferably 250 rpm.

6. The use of pichia pastoris, as claimed in claim 1, for the preparation of up-regulators of gene expression of zon-1, laminin and/or fibronectin; the laminin is preferably laminin 332.

7. The use of claim 6, wherein the Pichia pastoris is cultured to obtain a fermentation product thereof, and the gene expression up-regulator is prepared using the fermentation product.

8. Use according to claim 6 or 7, wherein the up-regulator of gene expression is suitable for use in cosmetics or skin care products.

9. A method for preparing up-regulator of gene expression of zon-1, laminin and/or fibronectin, which comprises culturing Pichia pastoris according to claim 1, obtaining its fermentation product, centrifuging and collecting the supernatant;

the laminin is preferably laminin 332.

10. The method according to claim 9, wherein the gene expression up-regulator is used in a cosmetic or skin care product.