Method for producing periplaneta americana allergen protein Per a 5 in baculovirus-insect expression system

An allergen protein, Periplaneta americana technology, applied in the field of genetic engineering, can solve problems such as side effects, hindering the detection of special allergens in patients, and poor curative effect

Inactive Publication Date: 2011-05-11
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, anaphylaxis extracts used for treatment are crude mixtures of protein and non-protein components isolated from natural sources that contain a large number of components that are not related to allergens, or contain almost no allergens that cause hypersensitivity in patients, Therefore, the effect is not good
3. The use of monoclonal antibody-based immunoassays to identify the main allergen content has made important progress in the standardization of anaphylaxis extracts. The disadvantage is that patients who are sensitive to a specific anaphylaxis extract must Treatment with the same complex mixture of all extract components can lead to side effects, and in terms of allergy diagnosis, this method of skin testing using whole anaphylaxis extracts prevents the identification of specific allergens to which the patient is sensitized. detection
At present, the natural and recombinant allergen Per a 5 protein has not been reported

Method used

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  • Method for producing periplaneta americana allergen protein Per a 5 in baculovirus-insect expression system
  • Method for producing periplaneta americana allergen protein Per a 5 in baculovirus-insect expression system
  • Method for producing periplaneta americana allergen protein Per a 5 in baculovirus-insect expression system

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Experimental program
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Embodiment 1

[0020] 1. The steps of this embodiment are as follows:

[0021] (1) Extract total RNA from Periplaneta americana tissue; take artificially raised frozen Periplaneta americana (from Jiangsu Provincial Center for Disease Control), grind it with liquid nitrogen, and add Trizol reagent at 100 mg tissue / mL Trizol reagent. Let stand for 10 minutes, then add chloroform at 300ul / mL, mix well, let stand for 10min, centrifuge at 12000g for 10min, discard the supernatant, and add isopropanol at 500ul / mL. Mix well, let stand for 10 minutes, centrifuge at 12000g for 10 minutes, discard the supernatant, and the sediment at the bottom of the tube is the total RNA of Periplaneta americana. Add 75% ethanol to suspend the precipitate, centrifuge at 12000 g for 10 min, and discard the supernatant. Dry at room temperature for 5 min, add ribonuclease-free double distilled water to dissolve.

[0022] (2) Using the extracted total RNA of Periplaneta americana as a template, the reverse transcripti...

Embodiment 2

[0027] The specific IgE binding activity of the expressed Per a 5 protein is determined:

[0028] The specific IgE binding activity of the Per a 5 protein produced by the baculovirus-insect expression system and Escherichia coli (prepared for our laboratory) was measured by indirect ELISA method. The Per a 5 protein produced by the baculovirus-insect expression system is the purified Per a 5 protein after the embodiment one step (4); the source of the Per a 5 protein produced by Escherichia coli is the embodiment one step ( 2) The resulting Periplaneta americana allergen Per a 5 gene shown in SEQ ID No: 1 is cloned into the pCold II plasmid, transformed Escherichia coli Origami (DE3), induced and expressed overnight with 0.5mMIPTG, and the thalline is collected , supernatant was collected by centrifugation after sonication, and purified by Nickel affinity column.

[0029] Dilute the Per a 5 protein produced by the baculovirus-insect expression system and Escherichia coli to 1...

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Abstract

The invention relates to a method for producing periplaneta americana allergen protein Per a 5, in particular to a method for producing periplaneta americana allergen protein Per a 5 in a baculovirus-insect expression system, belonging to the technical field of genetic engineering. In the invention, by adopting the baculovirus-insect expression system, the periplaneta americana allergen protein Per a 5 is produced, so as to avoid the problems that the Per a 5 allergen structure is affected in different expression systems (such as bacterium and saccharomycetes), provide an affinity chromatography method and solve the problem that the Per a 5 allergen purifying process is complex. The activity of the obtained Per a 5 protein by adopting the baculovirus-insect expression system is more than 1.6 times of the activity of the obtained Per a 5 protein by adopting colibacillus.

Description

technical field [0001] The invention relates to a method for producing Periplaneta americana allergen protein Per a5, in particular to a method for producing Periplaneta americana allergen protein Per a5 in a baculovirus-insect expression system, and belongs to the technical field of genetic engineering. Background technique [0002] Allergic diseases are one of the major human diseases. Its incidence rate is currently estimated to account for 30-40% of the world's population, and is increasing at a rate of more than 1% per year, with the most obvious increase in the incidence rate of children. my country's large-scale national surveys in recent years have shown that the incidence of asthma in children has increased significantly compared with 10 years ago, and there are as many as 10 million children with asthma, among which allergies are the main cause (National Children's Asthma Prevention and Control Cooperative Group 2003). In addition, unlike cancer and cardiovascular...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12P21/02
Inventor 何韶衡魏继福
Owner JIANGSU PROVINCE HOSPITAL
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