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Pichia pastoris and application thereof

A technology of yeast and seeds, applied in the field of microorganisms, can solve the problems of increased skin wrinkles, decreased expression levels, and lack of in-depth discussion and interpretation, and achieve the effect of increased growth vitality and enhanced expression levels

Pending Publication Date: 2020-10-20
JALA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the space environment can improve the expression level of exogenous genes in Pichia pastoris remains to be explored, and the biochemical and molecular biological mechanisms of space effects, including whether to induce genes in Pichia pastoris from transcription factors, regulatory proteins, etc. The differential expression of the
[0005] In addition, three proteins, claudin, laminin, and fibronectin, are expressed in the epidermis, dermal epidermal junction, and dermis of the skin, and with age, one or more of these three proteins are expressed decrease in volume, resulting in an increase in wrinkles on the skin
However, so far, there are few reports on substances that can simultaneously enhance the expression of these three proteins in the prior art

Method used

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  • Pichia pastoris and application thereof
  • Pichia pastoris and application thereof
  • Pichia pastoris and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Identification of the basic characteristics of aerospace yeast

[0037] 1. Medium

[0038] Seed medium (BMGY medium): 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34wt% YNB (amino-free yeast nitrogen source), 4×10 -5 wt % Biotin (Vitamin H) and 1 v / v % Glycerol, balance water.

[0039] Induction medium (BMMY medium): 2wt% peptone, 1wt% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34%wt YNB, 4×10 -5 wt% biotin (vitamin H) and 0.5v / v% methanol, balance water.

[0040] 2. Culture conditions

[0041] Cultivate in the seed medium at 30°C and 250rpm for 16-18 hours until OD 600 After 2 to 6, centrifuge at 4000rpm for 2min to obtain the thallus; inoculate the thalline with an inoculum size of 5% (volume fraction) in the induction medium at 30°C and 250rpm for 48 hours, and add 0.5v / v every 24h % methanol.

[0042] 3. Comparison of morphological features

[0043] After culturing SP02-CK-01 and SP02-DZ-08 on YPD agar ...

Embodiment 2

[0054] Example 2 Vitality comparison of aerospace yeast before and after loading

[0055] 1. Inoculate the bacterial cells stored in glycerol tubes of SP01-DZ-49, SP02-CK-01 and SP02-DZ-08 into 10ml liquid PDB medium at an inoculation amount of 1‰, and culture at 28°C and 200rpm for 24h;

[0056] 2. Take a sufficient amount of the three bacterial solutions, centrifuge at 4°C and 400rpm for 10 minutes to remove the supernatant, and resuspend in sterile saline to obtain a bacterial suspension. Dilute with normal saline, make different concentration gradients of the three bacterial suspensions in a 96-well plate (total volume 200 μL), record the OD of each bacterial suspension 600 ≈1.0 dilution ratio;

[0057] 3. According to the above quantitative dilution volume, add the three kinds of bacterial suspensions to 2.0mL PDB in the 12-well plate, keep the total volume 3.0mL unchanged, make up the rest with normal saline, and make 3 parallels for each strain;

[0058] 4. Record the...

Embodiment 3

[0063] Example 3 Preparation of Pichia pastoris fermentation product

[0064] 1. Medium preparation: Prepare seed medium and induction medium respectively according to the growth requirements of yeast SP02-DZ-08, sterilize at 115°C for 20 minutes under high temperature and high pressure, and cool down for later use.

[0065] 2. Strain activation: Pick the strains preserved at -80°C, put them into the liquid seed medium, and culture them in a shaker at 30°C and 250rpm for 16-18 hours to activate the strains.

[0066] 3. Purification of strains: Gradiently dilute and spread the activated bacterial solution in the previous step to obtain a single colony.

[0067] 4. Bacteria expansion culture: Pick a single colony from the plate in the previous step and inoculate it into the liquid BMGY medium, and culture it on a shaking table at 30°C and 250rpm for 16-18 hours to obtain the fermentation bacteria seed liquid.

[0068] 5. Yeast inoculation and fermentation: add the expanded yeas...

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Abstract

The invention discloses a strain of pichia pastoris and an application thereof. The preservation number of the pichia pastoris is CCTCC NO: M 2016763. Compared with the polypeptide expression quantityof homogeneous fungal strains in the prior art, the polypeptide expression quantity of a pichia pastoris fungal strain disclosed by the invention is substantially increased, and the increase range can reach 36.32%. In addition, the expression product of the pichia pastoris disclosed by the invention has obvious reinforcement effects on the expression quantity of tight junction proteins ZO-1, laminin and fibronectin, and can be used for preparing gene expression upregulation agents of the tight junction proteins ZO-1, the laminin and the fibronectin.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a Pichia pastoris and an application thereof. Background technique [0002] Space breeding mainly induces genetic variation of organisms through comprehensive space environmental factors such as strong radiation, microgravity and high vacuum. On the one hand, there are many factors that induce biological mutations in the space environment. Space mutation breeding is an effective breeding method that can greatly shorten the development cycle of new varieties. On the other hand, the space environment has an important impact on the morphological structure, growth rate, metabolic activity, and gene expression of microorganisms. Microorganisms are small in size, simple in culture conditions, and more likely to cause mutations than higher organisms. They are better materials for space biology research. [0003] Yeast is a large group of single-celled eukaryotic microorganism...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N1/38C12P21/02C07K14/78A61K8/9728A61Q19/00C12R1/84
CPCC12N1/38C12P21/02C07K14/78A61K8/9728A61Q19/00A61K2800/10C12R2001/84C12N1/165
Inventor 李俊李慧章漳蒋丹丹
Owner JALA GROUP CORPORATION