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Preparation method and application of restructured dust mite II type allergen Der p2 and Der f2 protein

An allergen and dust mite technology, which is applied in the field of preparation of recombinant type II allergen Derp2 and Derf2 proteins, can solve problems such as incorrect structure of DF2 protein, difficult to meet clinical dosage, weak serum reactivity, etc. Achieve the effects of increased yield, improved activity, and simple ingredients

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

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Problems solved by technology

At present, the research on the recombinant expression of DP2 using the eukaryotic expression system is more representative of the patent of Stallergenes in 2011 (European patent number: EP2388268 (A1)), they used the Pichia pastoris expression system to carry out recombinant expression and purification of DP2 , the patent does not optimize the DP2 gene and molecular construction for the Pichia pastoris system, the yield is low, the purification process is complicated, the yield is low, and it is difficult to meet the clinical dosage
The representative ones for the recombinant expression of DF2 are the research conducted by Hu Youying et al. in the prokaryotic expression system in 2011, and the "A method for producing recombinant D. farinae allergens Der f1 and Der f2 fusion proteins" filed by Cui Yubao et al. in 2012. (Chinese Patent No.: CN102676568A), but the prokaryotic expression system has no post-translational modification function, the obtained DF2 protein structure is incorrect, and the reactivity with serum is weak, and it is difficult to separate and purify later

Method used

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  • Preparation method and application of restructured dust mite II type allergen Der p2 and Der f2 protein
  • Preparation method and application of restructured dust mite II type allergen Der p2 and Der f2 protein
  • Preparation method and application of restructured dust mite II type allergen Der p2 and Der f2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 recombinant DP2 / DF2 small-scale (3L) high-density fermentation process

[0057] Step 1: Activation of recombinant strains

[0058] frozen at -80°C DP2 Glycerol bacteria seeds in the working seed bank were streaked on YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L), and cultured in a constant temperature and humidity box at 30°C for 3-5 days .

[0059] Step 2: Seed liquid culture

[0060] Pick the monoclonal colonies on the solid medium 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.

[0061] Step 3: Fermentation Process

[0062] 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 med...

Embodiment 2

[0068] Example 2 Recombinant DP2 / DF2 large-scale (30L) high-density fermentation verification

[0069] Step 1: Activation of recombinant strains

[0070] 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.

[0071] Step 2: Primary seed liquid culture

[0072] Pick the monoclonal colony on the solid medium in step 1 and put it in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), 30°C, 220rpm and cultivate to OD 600 ≈6.0, and observed under a microscope without any bacteria, the first-grade seed liquid for fermentation was obtained.

[0073] Step 3: Secondary Seed Solution Culture

[0074] 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 dissolved oxygen...

Embodiment 3

[0079] Example 3 Recombinant DP2 / DF2 Pilot Test (300L) High Density Fermentation Verification

[0080] Step 1: Activation of recombinant strains

[0081] 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.

[0082] Step 2: Primary seed liquid culture

[0083] Pick the monoclonal colony on the solid medium in step 1 and put it in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), 30°C, 220rpm and cultivate to OD 600 ≈6.0, and observed under a microscope without any bacteria, the first-grade seed liquid for fermentation was obtained.

[0084] Step 3: Secondary Seed Solution Culture

[0085] 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, and 300rpm, and dissolve it throu...

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Abstract

The invention relates to an industrial production method of restructured dust mite II type allergen Der p2 and Der f2 protein. Fermentation parameters are optimized, and a specific culture and fermentation control condition is adopted. Compared with a fermentation condition which is not optimized, the activity of fermentation products is improved by 50%, the yield is increased by 20% or above, theused fermentation culture medium is simple in components and low in cost, and the cost of a fermentation process is reduced. A purification process is optimized, so that the yield is increased by 30%. Host remaining protein and remaining DNA completely meet medical level requirements. The expressed restructured II type allergen Der p2 and Der f2 protein and the making method thereof have industrial application prospects.

Description

technical field [0001] The invention belongs to the field of bioengineering genes, and relates to a preparation method and application of recombinant type II allergen Der p2 and Der f2 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, and it is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/435C07K1/18C07K1/20C12R1/84
CPCY02A50/30
Inventor 马永范宇张韬
Owner ZONHON BIOPHARMA INST
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