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Preparation of recombinant dust mite type I allergen Der p1 and Der f1 protein and application

A technology for allergens and dust mites, applied in the field of industrialized production of recombinant type I allergen Derp1 and Derf1 proteins, can solve the problems of DP1 and DF1 protein expression and fermentation process optimization, unsuitable for large-scale preparation, mature protein harvesting. problems such as low yield, to achieve the effect of increased yield, simple ingredients and high activity

Active Publication Date: 2019-06-07
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most comprehensive research on the recombinant type I dust mite allergen (Der P1, hereinafter referred to as DP1) and recombinant type I dust mite allergen (Der f1, hereinafter referred to as DF1) was carried out by Japanese scholar Toshiro Takai in 2005. The article shows that when DP1 and DF1 are expressed in the Pichia pastoris system, protein propeptides need to be added, otherwise they cannot be expressed in the eukaryotic expression system, and then undergo an activation process to obtain mature DP1 and DF1 that are consistent with the amino acid sequence of the natural protein. DF1 protein, this article studies the activation method of DP1 and DF1 protein and the protein activity before and after activation, and proves that the activity of the activated protein is consistent with that of the natural protein, which is higher than that of the pre-activated protein; but the method introduced in the article has a low yield of mature protein , is not suitable for large-scale production, and has not optimized the expression and fermentation process of DP1 and DF1 proteins; besides, there is no further research report for the time being

Method used

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  • Preparation of recombinant dust mite type I allergen Der p1 and Der f1 protein and application
  • Preparation of recombinant dust mite type I allergen Der p1 and Der f1 protein and application
  • Preparation of recombinant dust mite type I allergen Der p1 and Der f1 protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 recombinant DP1 / DF1 small-scale (3L) high-density fermentation process

[0059] Step 1: Activation of recombinant strains

[0060] Streak the Glycerol bacteria seeds in the DP1 working seed bank frozen at -80°C on the YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L), 30°C Cultivate in a constant temperature and humidity box for 3-5 days.

[0061] Step 2: Seed liquid culture

[0062] Pick the monoclonal colonies on the plate and culture them in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L) at 30°C and 220rpm until OD 600 ≈6.0, and observe under the microscope that there is no miscellaneous bacteria, and the seed liquid for fermentation is obtained.

[0063] Step 3: Fermentation Process

[0064] Clean the Sartorius B-plus bioreactor, calibrate the pH meter probes of the fermenter with pH=7.0 and pH=4.0 standard solutions, and then configure BSM medium as the fermentation medium (see the ta...

Embodiment 2

[0070] Example 2 Recombinant DP1 / DF1 large-scale (30L) high-density fermentation verification

[0071] Step 1: Activation of recombinant strains

[0072] Streak the Glycerol bacteria seeds in the working seed bank frozen at -80°C on YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L) and keep the temperature at 30°C Cultivate in a humid chamber for 3-5 days.

[0073] Step 2: Primary seed liquid culture

[0074] Pick the monoclonal colonies on the plate in step 1 and culture them in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L) at 30°C and 220rpm to OD 600 ≈6.0, and observed under a microscope without any bacteria, the first-grade seed liquid for fermentation was obtained.

[0075] Step 3: Secondary Seed Solution Culture

[0076] Inoculate the primary seed liquid obtained in step 2 into the Sartorius B-plus fermenter, and cultivate it with 60% BSM, pH=5.5±0.2, 27°C, 300rpm, and maintain the dissolved oxygen thr...

Embodiment 3

[0081] Example 3 Recombinant DP1 / DF1 Pilot Test (300L) High Density Fermentation Verification

[0082] Step 1: Activation of recombinant strains

[0083] Streak the seeds of glycerol bacteria in the working seed bank frozen at -80°C on YPD slant medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L), and keep the temperature at 30°C Cultivate in a humid chamber for 3-5 days.

[0084] Step 2: Primary seed liquid culture

[0085] Pick the monoclonal colonies on the plate in step 1 and culture them in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L) at 30°C and 220rpm to OD 600 ≈6.0, and observed under a microscope without any bacteria, the first-grade seed liquid for fermentation was obtained.

[0086] Step 3: Secondary Seed Solution Culture

[0087] Inoculate the primary seed solution obtained in step 2 into a Diwoxin BVT-3000 30L seed tank, and cultivate it with 60% BSM, pH=5.5±0.2, 27°C, 300rpm, and make the solution Oxygen...

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Abstract

The invention relates to a preparation method of recombinant dust mite type I allergen Der p1 and Der f1 protein. Fermentation parameters are optimized, and specific culture and fermentation control conditions are adopted, so that compared with the fermentation condition which is not optimized, the activity of a fermentation product is improved by 50%, and the yield is increased by 20%; besides, used fermentation mediums are simple in ingredients and low in cost, and the cost of a fermentation technology is reduced; and a purification technology is optimized, so that the yield is increased by30%. Host remain protein and remaining DNA can completely meet medical level requirements, which indicates that the recombinant type I allergen Der p1 and Der f1 protein and the preparation method thereof have industrialization application prospects.

Description

technical field [0001] The invention belongs to the field of bioengineering genes, and relates to an industrial production method and application of recombinant type I allergen Der p1 and Der f1 proteins. Background technique [0002] There are many kinds of dust mites, which widely exist in human living and working environments. Their excreta, metabolites and mite bodies all have strong allergenicity. According to statistics, about 10% of the world's population is allergic to dust mites, and about 80% of Extrinsic asthma is caused by dust mites. [0003] At present, the crude extract of dust mite allergens is mainly used clinically to treat allergic diseases caused by dust mites. extract. Dust mite allergens mainly exist in excreta and mites, and the extraction method takes a long time, the process is cumbersome, and the cost is high; in addition, the composition of the natural allergen extract is very complicated, and it is very difficult to keep its components constant,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/435C07K1/18C07K1/20
CPCY02A50/30
Inventor 马永范宇张韬
Owner ZONHON BIOPHARMA INST
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