Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein

A fusion protein and Dermatophagoides farinae technology, applied in the field of producing recombinant Dermatophagoides farinae allergens Der f1 and Der f2 fusion proteins, can solve the side effects of immunotherapy that cannot fundamentally improve the purity of allergens, cannot completely remove solvents, process Cumbersome and other problems, to achieve the effect of low cost, avoid side reactions, and simple process

Inactive Publication Date: 2012-09-19
崔玉宝 +3
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Problems solved by technology

[0004] Therefore, 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 infusions. However, dust mite allergens mainly exist in excreted The use of biochemical methods to purify dust mite allergens from food and leather shells is time-consuming, cumbersome, and costly, and it is impossible to completely remove solvents and other components, w

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  • Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein
  • Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein
  • Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein

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Embodiment

[0016] The plasmids, strains, reagents and instruments used for the cloning and expression of the full-length gene of the first component of D. farinae allergen were BamH I enzyme, Xho I enzyme, RNAiso Reagent (Code No. D312), One Step RNA PCR Kit ( Code No.DRR024A), PrimeSTARTM HS DNA Polymerase(Code No.DR010A), Agarose Gel DNA Purification Kit Ver.2.0(Code No.DV805), DNA A-Tailing Kit(Code No.D404), DNA Fragment Purification Kit Ver. 2.0 (Code No.DV807), DNA Ligation Kit (Code No.D6023), pMD19-T simple (Code No.D104), E.coli Competent Cells JM109 (Code No.D9052) were purchased from TaKaRa Company; pET28a (+ ) (Kit Lot No.N72770), TP3000 PCR instrument (TaKaRa BIO INC.), Mupid electrophoresis instrument (ADVANCE-BIO Co., Ltd), VDS electrophoretic imaging device (Pharmacia Biotech);

[0017] Cloning and sequencing of dust mite main allergen Der f 1: a. Design and synthesis of primers: design primers according to the sequence published by GenBank AB034946, and add BamH I / XhoI...

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Abstract

The invention discloses a method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein and relates to the technical field of construction of dermatophagoides farinae allergen gene by a biological technology. The method comprises the following steps of: obtaining a coding gene by extracting the total RNA of the dermatophagoides farinae and adopting PCR (Polymerase Chain Reaction) amplification, cutting glue to recover a product, named as Der fm, cloning to pMD19-T vectors, subcloning to expression vectors pET-28a(+), converting the vectors to escherichia coli, carrying out inducible expression by using IPTG (isopropyl-Beta-d-thiogalactoside). The method disclosed by the invention has the advantages that the consumed time is short, the process is simple, the cost is lower, the components such as a solvent can be thoroughly removed, the purity of the allergen can be increased fundamentally, the occurrence of side reaction in immunological therapy is avoided, the purity is high, the classification is clear, the accuracy of diagnosis can be increased, and a standard product is expected to be provided for novel immunological therapy.

Description

Technical field: [0001] The invention relates to the technical field of constructing dust mite allergens by biological technology, in particular to a method for producing fusion proteins of recombinant dust mite allergens Der f1 and Der f2. Background technique: [0002] There are many kinds of dust mites, which widely exist in the living and working environment of human beings. Their excreta, metabolites and mite bodies all have strong allergenicity. It is estimated that about 10% of the world's population is allergic to dust mites, and about 80% % of exogenous asthma is caused by dust mites. Scholars in my country used the crude extract of dust mite to conduct skin prick test on patients with allergic diseases. 47-92.11% of adult asthmatic patients, 51.64-78.85% of asthmatic children, 80% of asthmatic patients About 70% of patients with rhinitis and about 70% of patients with chronic urticaria were positive. In 1998, the direct and indirect economic losses caused by asthma i...

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Application Information

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IPC IPC(8): C12N15/70C12R1/19
Inventor 崔玉宝蔡红星杨庆贵王运刚
Owner 崔玉宝
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