Recombinant mite allergen protein drug mixture and application thereof

A protein drug and allergen technology, which is applied in the direction of drug combination, allergen antigen components, peptide/protein components, etc., can solve the problems of time-consuming, cumbersome process, and pathogen microbial contamination, so as to reduce antigenic epitopes, reduce Anaphylaxis, good immunogenic effect

Pending Publication Date: 2019-12-31
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the complex composition of the natural allergen extract, it is very difficult to keep its composition constant, and it is easily contaminated by exogenous toxic substances and pathogenic microorganisms, which affects its repeatability and safety. Local reactions such as flushing, swelling, induration, and necrosis, and systemic reactions such as shock, edema, bronchospasm, urticaria, angioedema, and generalized erythema. In addition, the use of crude extracts for diagnosis cannot clarify the patient’s response to allergens in each group. The degree of response can easily lead to misdiagnosis
The quality of allergens is crucial to the diagnosis and treatment of allergic diseases. The allergens used for immunodiagnosis and treatment should be pure products rather than crude extracts, and dust mite allergens mainly exist in excreta and mites Among them, the extraction method takes a long time, the process is cumbersome, the cost is high, and the occurrence of side effects in immunotherapy cannot be avoided

Method used

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  • Recombinant mite allergen protein drug mixture and application thereof
  • Recombinant mite allergen protein drug mixture and application thereof
  • Recombinant mite allergen protein drug mixture and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Recombinant proDP1 codon optimization transformation, construction of expression plasmid and construction of host engineering strain

[0052] Step 1: Recombination codon optimization transformation

[0053] According to the DNA sequence of DP1 published by EMBL-EBI (ENA accession number: FM177224.1), the inventor obtained the proDP1 gene of the present invention (the proDP1 gene (the The gene has an alpha-factor signal peptide and a Kozak sequence GCCACCATGG added on the basis of the wild-type gene, and has been codon-optimized), and the nucleotide sequence is shown in SEQ ID No:1.

[0054] According to the DNA sequence of DF1 published by GenBank (GenBank accession number: AB034946.1), the inventor obtained the proDF1 gene of the present invention after combining the requirements of the expression system of the present invention and multiple verifications, and obtained the proDF1 gene of the present invention (the gene is in The alpha-factor signal pepti...

Embodiment 2

[0069] Example 2 Recombinant DP1 / DF1 large-scale (30L) high-density fermentation experiment

[0070] The following will first illustrate with DP1 large-scale (30L) high-density fermentation

[0071] Step 1: Activation of recombinant strains

[0072] Get the glycerol bacteria seeds produced after the above-mentioned examples and freeze them in the working seed bank at -80°C in YPD solid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L) Streak the line and cultivate in a constant temperature and humidity chamber at 30°C 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 until 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 t...

Embodiment 3

[0081] Embodiment 3 recombinant DP1 / DF1 purification process

[0082] 1. The first step of purification of recombinant DP1 / DF1 fermentation broth supernatant

[0083] Step 1: Pretreatment of the fermentation broth

[0084] The fermentation broth obtained in the above examples was centrifuged at high speed to obtain a supernatant; diatomaceous earth was added to aid in filtration, and a clarified fermentation broth sample was obtained overnight; diluted with a 10KD membrane bag ultrafiltration to reduce the conductance to below 5mS / cm.

[0085] Step 2: Cation Exchange Chromatography

[0086] The acetic acid of the fermented liquid supernatant after the above treatment is adjusted to pH 4.0, and put on the SP FF chromatography column. 50mM NaAc, 1.0M NaCl, pH4.0, according to 0-100% linear elution, DP1 / DF1 target protein is mainly concentrated in the second elution peak.

[0087] The rest of the conditions remained unchanged, and the pH of the sample and buffer was adjust...

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Abstract

The invention relates to a recombinant mite allergen protein composition, the recombinant mite allergen protein composition comprises recombinant mite I-type allergens DP1 and DF1 protein and recombinant mite II-type allergens DP2 and DF2 protein which are respectively mixed according to the activity ratio of 1: 1: 1: 1 or mixed according to the mass ratio of (1-5): (1-20): (1-10): (1-20). Compared with an existing product, the protein composition has the advantages that (1) the recombinant allergen has high purity, controllable quality and the like; (2) the recombinant protein has good specificity; and (3) compared with a natural extracting solution, the protein composition has better immunogenicity, the risk of serious allergic side reactions is reduced, and the desensitization treatmenteffect is improved; secondly, the recombinant allergen composition disclosed by the invention not only has better activity than natural allergen protein in the same proportion, but also has activityhigher than that of a corresponding natural single component compared with that of a single component, and has a product development and application prospect.

Description

technical field [0001] The invention relates to a drug combination for treating dust mite allergy, in particular to an improved recombinant dust mite allergen protein drug mixture and application thereof. Background technique [0002] Among the many allergens that cause allergic diseases, dust mites are the most important allergens. The positive rate of dust mites in specific immunodiagnosis of patients with allergic diseases is about 70-80%. There are many kinds of dust mites, which widely exist in human living and working environments. Their excrement, metabolites and mite bodies all have strong allergies. According to statistics, about 10% of the world's population is allergic to dust mites, and about 80% of foreign Allergic asthma is caused by dust mites. [0003] At present, dust mite allergen extracts are mainly used clinically to treat dust mite allergies. For example, the "Changdi" dust mite drops of Zhejiang Wowu Biology, which was launched in 2006, is the extract...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P37/08A61P33/14C12N15/81C12P21/02C07K14/435C07K1/18C07K1/20C12R1/84
CPCA61K38/1767A61P37/08A61P33/14C12N15/815C07K14/43531A61K2300/00C12P21/02A61K39/35C12N1/16C07K2319/02
Inventor 马永范宇吴诚赵利利江辰阳
Owner ZONHON BIOPHARMA INST
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