A method for promoting the secretion and expression of human beta-defensin 3 by Saccharomyces cerevisiae by using Polygonum water

A secreted expression, Saccharomyces cerevisiae technology, applied in the field of microbial applications, can solve problems such as affecting the correct folding of proteins, many miscellaneous proteins, and restricting applications, and achieve the effects of promoting growth, increasing protein content, and promoting obvious effects.

Active Publication Date: 2017-04-05
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the Escherichia coli expression system has certain simplicity and popularity, there are still some defects: 1) lack of post-translational modification and processing of eukaryotic proteins, and disulfide bonds cannot be formed correctly, which affects the correct folding of proteins. 2) Most of the expressed proteins exist in the form of inclusion bodies, and the purification and recovery of defensins will not only increase the cost, but also affect the biological activity of defensins; 3) There are many miscellaneous proteins, and the purification steps are complicated, etc.
However, as a fungal expression system, Saccharomyces cerevisiae has low protein expression, which limits its application.

Method used

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  • A method for promoting the secretion and expression of human beta-defensin 3 by Saccharomyces cerevisiae by using Polygonum water
  • A method for promoting the secretion and expression of human beta-defensin 3 by Saccharomyces cerevisiae by using Polygonum water
  • A method for promoting the secretion and expression of human beta-defensin 3 by Saccharomyces cerevisiae by using Polygonum water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Preparation of seed liquid:

[0053] Streak the preserved recombinant Saccharomyces cerevisiae INVSC1 / pYα-hBD-3 glycerol strain on the YPD medium plate, place it in a 30°C incubator and cultivate it for 2-3 days until a single colony grows;

[0054] The formula of the YPD medium used is as follows: glucose 20g / L, yeast extract 10g / L, peptone 20g / L; agar 15g / L was added to the solid medium.

[0055] Pick a plump single colony from the above-mentioned single colony and inoculate it on a YPD medium plate, culture it in a 30°C incubator for 2 days, then pick a plump single colony and inoculate it in 5-10mL YPD liquid medium, at 30°C , 250r / min shaking flask culture overnight, to obtain seed liquid.

[0056] (2) Inoculate 1 mL of the seed liquid prepared in step (1) into 100 mL of sterilized Polygonum water growth medium, culture at 30°C with shaking at 250 r / min, and culture for 24 hours to OD 600 Stop growth and culture at 1 to 4 o'clock to obtain a culture solution....

Embodiment 2

[0065] (1) Take 1 mL of the seed liquid prepared in Example 1-(1) and inoculate it into 100 mL of sterilized Polygonum water growth medium, cultivate it with shaking at 29°C and 220 r / min, and cultivate it for 30 hours to OD 600 Stop growth and culture at 1 to 4 o'clock to obtain a culture solution.

[0066] The formula of the Polygonum water growth medium used is as follows: 70g / L glucose, 6.7g / L YNB, 0.1 each of adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan g / L, aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine and valine each 0.05g / L, 30g / L Peptone, 40g / L Polygonum water.

[0067] (2) Take the culture medium in step (1) and centrifuge at low speed (5000rpm, 10min), discard the supernatant, add 10mL sterile water to wash twice, centrifuge at low speed (5000rpm, 10min), discard the supernatant, and obtain the recombinant wine Yeast growth culture cells.

[0068] (3) Transfer the recombinant Saccharomyces cer...

Embodiment 3

[0074] (1) Inoculate 1 mL of the seed liquid prepared in Example 1-(1) into 100 mL of sterilized Polygonum water growth medium, culture at 28°C with shaking at 180 r / min, and culture for 36 hours to OD 600 Stop growth and culture at 1 to 4 o'clock to obtain a culture solution.

[0075] The formula of the growth medium of Polygonum waterus used is as follows: 90g / L glucose, 6.7g / L YNB, 0.1 each of adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan g / L, aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine and valine each 0.05g / L, 40g / L Peptone, 60g / L Polygonum water.

[0076] (2) Take the culture medium in step (1) and centrifuge at low speed (5000rpm, 10min), discard the supernatant, add 10mL sterile water to wash twice, centrifuge at low speed (5000rpm, 10min), discard the supernatant, and obtain the recombinant wine Yeast growth culture cells.

[0077] (3) Transfer the recombinant Saccharomyces cerevisiae growth cul...

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Abstract

The invention discloses a method for utilizing polygonum hydropiper to promote saccharomyces cerevisiae to secrete and express human beta-defensin-3 (hBD-3). The method comprises the following steps: preparing a strain liquid of recombination saccharomyces cerevisiae INVSC1 / pY alpha-hBD-3; inoculating the strain liquid to a growth medium of polygonum hydropiper for culturing, and collecting thallus; inoculating the thallus to an induction medium of polygonum hydropiper for culturing, and collecting the supernatant; and performing a bacteriostatic test, and using the diameter of an inhibition zone to represent the bacteriostatic effect. According to the technical scheme, the growth medium and the induction medium, which are novel, low in cost and high in secretion amount, are provided, and the capability of recombination saccharomyces cerevisiae INVSC1 / pY alpha-hBD-3 on secreting and expressing human beta-defensin-3 is improved. The matching of a carbon-nitrogen ratio in the growth medium and the induction medium are extremely beneficial to growth of saccharomyces cerevisiae and secretion and expression of protein, the method is obviously more excellent than conventional culturing methods, and provides a relatively firm foundation for industrialization of recombination saccharomyces cerevisiae.

Description

technical field [0001] The invention belongs to the field of microbe application, and in particular relates to a method for promoting the secretion and expression of human beta defensin 3 by Polygonum hydroponica. Background technique [0002] Defensins are the largest subfamily of the antimicrobial peptide family. They are a type of defensive polypeptide active substances produced in vivo against the pathogenic effects of exogenous pathogens. They are an important part of the innate immunity of organisms. Consists of the host's immune defense system. So far, the research has found that the types of defensins are: mammalian defensins, insect defensins, and plant defensins; and according to the position of the disulfide bond, the difference in connection, the difference in precursors and expression methods, defensins are divided into α- There are three types of defensins, β-defensins and θ-defensins. [0003] Human β-defensin 3 (human D-defensin-3, hBD-3) is the peptide wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/865
Inventor 唐语谦刘思利钟凤吴晖
Owner SOUTH CHINA UNIV OF TECH
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