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Recombinant fusion alkaline phosphatase-allergen protein and preparation method and application thereof

An allergen protein and phosphatase technology, applied in biochemical equipment and methods, ovalbumin, transferrin, etc., can solve the problems of complex glycosylation modification function, inability to recombine protein modification, and difficult protein renaturation, etc. To achieve the effect of reducing non-specific reactions, improving detection sensitivity, and strong specificity

Active Publication Date: 2018-07-03
GUANGZHOU INST OF RESPIRATORY DISEASE +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The expression methods of recombinant allergens are mainly divided into prokaryotic expression, yeast expression, insect cell, mammalian cell expression, and plant cell expression. Prokaryotic expression has a high yield, but some necessary modifications can not be made to the recombinant protein. protein refolding
Using eukaryotic cells to express allergens, the protein can be folded correctly and can provide complex and accurate glycosylation modification functions. Therefore, the expression products are closest to natural higher protein molecules in terms of molecular structure, physical and chemical properties and biological functions, but relatively Generally speaking, the expression yield is low, purification is difficult, biotinylation labeling can be carried out only when the protein concentration is high, and purification is required after labeling to remove unlabeled substances, etc. In general, the operation is complicated and the cost is high

Method used

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  • Recombinant fusion alkaline phosphatase-allergen protein and preparation method and application thereof
  • Recombinant fusion alkaline phosphatase-allergen protein and preparation method and application thereof
  • Recombinant fusion alkaline phosphatase-allergen protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of Alkaline Phosphatase Fusion Allergen Recombinant Plasmid

[0038] 1. Preparation method of allergen gene:

[0039] 1.1 Find the amino acid sequences of crustacean TM, crab CAK, egg Gald1, egg Gald2, egg Gald3, and cockroach Blag5 and Blag8 from the NCBI database, and use CODEHOP to design merger primers for each species.

[0040] 1.2 Tissue preparation

[0041] (1) Buy fresh shrimp from the vegetable market, cut the muscle into small pieces, and store them at -80°C for later use.

[0042] (2) Remove the shells of the bought live crabs, scrape the muscles with a knife, cut into small pieces, and freeze them at -80°C for later use.

[0043] (3) Purchase chicken oviducts freshly taken out of the chicken from the vegetable market, cut them into pieces with scissors, and store them frozen at -80°C for later use.

[0044] (4) German cockroaches caught indoors were starved overnight, cut into pieces, and frozen at -80°C for later use.

[0045] 1.3 G...

Embodiment 2

[0064] Example 2: Construction of CHO stable cell lines expressing allergens

[0065] In this embodiment, the fusion expression of alkaline phosphatase-allergen is carried out in mammalian cell CHO. In other embodiments, host cells are selected from insect cells, yeast and plant cells. In this example, a CHO stable cell line expressing allergens was constructed.

[0066] 1. Culture medium, reagents, equipment, etc. used for cell line construction:

[0067] Cell: CHO;

[0068] Medium: CHO serum-free medium, fetal bovine serum, double antibody;

[0069] Freezing medium: fetal bovine serum or newborn bovine serum containing 10% DMSO

[0070] Antibiotics: Puromycin;

[0071] trypsin;

[0072] Transfection kit lipofectamine2000;

[0073] CO 2 incubator;

[0074] Ultra-clean workbench;

[0075] Microscope etc.

[0076] 2. Stable cell line construction steps

[0077] 2.1 Puromycin (puromycin) concentration screening

[0078] (1) Well-grown CHO cells are seeded into 24-wel...

Embodiment 3

[0099] Embodiment 3: Alkaline phosphatase fusion allergen recombinant protein production

[0100] Media, reagents, equipment, etc. used in the production of allergen recombinant proteins:

[0101] Medium: CHO serum-free medium;

[0102] Equipment: shaker bottle, shaker;

[0103] Allergen recombinant protein fermentation steps are as follows:

[0104] 1. Resuscitated and screened stable cell line CHO;

[0105] 2. Subculture to 4ml medium, grow for about 3 days, and identify by ELISA;

[0106] 3. Digest the cells, subculture to 10ml medium, culture for 2-3 days, add 10ml medium, make the cell density not exceed 1*10 6 a / ml;

[0107] 4. Subculture 20ml of cells into a 250ml large bottle, continue to cultivate, and add fresh medium continuously during the period, so that the cell density does not exceed 1*10 6 cells / ml, so that the volume of cell fluid does not exceed 1 / 3 of the bottle capacity;

[0108]5. Change the 500ml culture bottle and repeat 4.

[0109] 6. Collect t...

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Abstract

The invention provides recombinant fusion alkaline phosphatase-allergen protein and a preparation method and application thereof, and comprises an expression vector of recombinant fusion alkaline phosphatase-allergen protein, a preparation method of the recombinant fusion alkaline phosphatase-allergen protein, the recombinant fusion alkaline phosphatase-allergen protein and application of the recombinant fusion alkaline phosphatase-allergen protein in in-vitro diagnosis of I type allergy. The invention also provides a kit and a detection method for detecting allergen-specific IgE antibody. Theprotein structure and physical and chemical properties of the recombinant fusion alkaline phosphatase-allergen protein are closer to natural allergen protein, and therefore, detection sensitivity ishigh and non-specificity is less; conventional mark inter-assay difference is avoided absolutely, and the production process of markers is simplified; the preparation process of key materials is simplified, production cost is lowered, the activity of allergen is improved, and kit use operation step and time are saved.

Description

technical field [0001] The invention relates to the technical field of in vitro immunodiagnosis, in particular to a recombinant fusion alkaline phosphatase-allergen protein and its preparation method and application, in particular to a recombinant fusion alkaline phosphatase-allergen protein IgE in vitro detection kit Applications. Background technique [0002] Allergies are mostly IgE-mediated adverse immune responses to antigen molecules in nature. Under normal circumstances, IgE specific to an allergen cannot be detected in the blood, and it is only produced after the human body is sensitized by external antigenic substances. Allergens, the substances that cause allergic reactions, induce the production of a specific type of IgE that reacts only with that substance. sIgE (specific IgE, specific IgE) combines with allergens to cause cells to release histamine, and histamine triggers allergies, such as asthma, eczema, rash, itchy eyes, rhinitis, runny nose, etc., and even...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/85C07K19/00C12N9/16C12N9/12G01N33/68G01N33/573
CPCC07K14/465C07K14/4716C07K14/4717C07K14/4732C07K14/76C07K14/77C07K14/79C07K2319/00C12N9/1223C12N9/16C12N9/2462C12N15/85C12Y207/03003C12Y301/03001C12Y302/01017G01N33/573G01N33/68G01N2333/415G01N2333/43582G01N2333/46G01N2333/4712G01N2333/4713G01N2333/76G01N2333/9123G01N2333/936
Inventor 孙宝清罗文婷李晨阳李小锋李园枚吴慧洁郑佩燕黄惠敏韦妮莉黄金玲
Owner GUANGZHOU INST OF RESPIRATORY DISEASE
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