CN102455362A

CN102455362A - Standard-antigen-based ELISA detection method of peanut allergenic protein Arah2

Info

Publication number: CN102455362A
Application number: CN2010105144345A
Authority: CN
Inventors: 陈红兵; 吴志华; 胡纯秋; 杨安树; 李欣; 高金燕
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2010-10-21
Filing date: 2010-10-21
Publication date: 2012-05-16

Abstract

The invention relates to a standard-antigen-based ELISA detection method of a peanut allergenic protein Arah2. The method comprises steps that: the DNA sequence of a standard antigen is acquired; the standard antigen is expressed and purified, a recombination protein polyclonal antibody is prepared, and a competitive inhibition ELISA system based on the standard antigen is established. The method is characterized in that: the obtained standard substance is a recombination expressed Arah2 protein, and the protein is used as an antigen for preparing an antibody required in ELISA detection. According to the invention, the recombined peanut allergenic protein Arah2 is adopted as a standard antigen, the standard antibody is prepared, and the standard-antigen-based ELISA detection method of the peanut allergenic protein Arah2 is established on the basis. The recombined food allergen can be produced with large quantities, and has advantages such as high purity and stable quality. The allergen can be used for replacing natural extracts in allergen ELISA detections, and provides high sensitivity and specificity. With the method provided by the invention, standardization of qualitative and quantitative detection reagents can be realized.

Description

ELISA detection method based on the peanut allergen protein Arah2 of standard antigen

Technical field

The invention belongs to the food inspection technical field, particularly the detection technique of allergen protein.

Background technology

Enzyme linked immunological absorption detects (Enzyme-linked immunosorbent assay; ELISA) be a kind of enzyme immunization method; The elisa technique development of phase late 1990s rapidly; In addition the application of full-automatic enzyme micro-plate reader is greatly improved the specificity of ELISA and sensitivity, expands greatly in the food inspection Application for Field.The characteristics of ELISA are immobilization and enzyme labelings of antigen or antibody, and at present the most frequently used ELISA, antibody will be combined in the surface and sample protein (antigen) free responding of solid phase carrier.Detect reaction bonded at the sample protein (antigen) of surface of solid phase carriers with other a kind of antibody (two anti-) behind antibody and the antigen-reactive with enzyme labeling.Be combined in antibody one timing of surface of solid phase carriers, the sample protein (antigen) that combines with it is many more, and the color reaction of the antibody of enzyme labeling is just strong more, and the concentration of serum antibody is high more simultaneously, and the ELISA colour developing is also strong more.Biotin labeled two anti-and contain the Streptavidin of peroxidase in order to have used among the food allergen ELISA that can detect trace.

Elisa technique has been widely used in the fast detecting analysis of residue of veterinary drug, residues of pesticides, eqpidemic disease, harmful microorganism, animals and plants toxin etc. aspect food security, and has obtained effect preferably.Along with people's has developed the qualitative or quantitative detecting analysis that multiple ELISA method is used for food allergen now to the reinforcement of the deep and attention degree of food irritability understanding.ELISA has become a kind of seriation, traceization, commercial food security method for quick, is one of present most widely used Measurement for Biotechnique.It is the method for quick of anaphylactogen that European Union, the U.S., Canada and the Japan and other countries of having implemented the anaphylactogen statutory standard also all recommended the ELISA method.

Anaphylactogen detects in the used kit at present, and the allergen protein that adopts separation and purification as antigen preparation antibody more, and as standard items, the allergen protein of treating in the sample article carries out qualitative or detection by quantitative.Because of food allergen is (sugar) albumen, some is stable for this type (sugar) albumen, some instability, and this is restricted with regard to the susceptibility that makes test.Even anaphylactogen is enough stable, the different of its source and separation and purification approach also can cause the differences of this protein so that influence the quality of corresponding specific antibody, finally cause the detectable can't standardization.

Summary of the invention:

The objective of the invention is to set up a kind of peanut allergen protein Ara h2 ELISA detection method based on standardization antigen.High sensitivity, the high specific of realizing this albumen detect, and improve quantitative result stability and repeated, make that different batches sample quantitative result is comparable.

The ELISA detection method of peanut allergen protein Ara h2 based on standard antigen of the present invention mainly comprises the following steps:

1, the dna sequence dna of standard antigen obtains

Extract the total RNA of peanut.

Adopt the RT-PCP technology, with the RNA reverse transcription of coding peanut Ara h2 albumen, the double-stranded DNA that acquisition is corresponding with it also increases.

2, standard antigen, the expression and purification of the peanut Ara h2 albumen of promptly recombinating

The dna sequence dna that a last step is obtained is connected on the appropriate carriers.

To connect product is transformed in the competent cell.

Induce target gene in cell, to express.

The separation and purification target protein, and carry out purity and concentration evaluation.

3, recombinant protein Polyclonal Antibody Preparation

Cut glue and reclaim pure allergen protein of electrophoresis and emulsification.

The immunity new zealand white rabbit prepares polyclonal antibody.

Separate antiserum and carry out titration.

4, the competition based on standard antigen suppresses the foundation of ELISA system

Confirm envelope antigen and sero-fast dilutability

Competition suppresses the foundation of ELISA typical curve

The present invention adopts reorganization peanut allergen protein Ara h2 as standard antigen, preparation standard antibody, and set up peanut allergen protein Ara h2 ELISA detection method on this basis based on standardization antigen.Advantages such as high-purity, stay in grade can produced and have to the reorganization food allergen in a large number, and alternative natural extract is used for anaphylactogen ELISA and detects, and sensitivity is higher, specificity is stronger, can realize qualitative and standardization detection by quantitative reagent.

Description of drawings

Fig. 1 is a RT-PCR product electrophoresis result.

Fig. 2 is a recombinant plasmid bacterium colony PCR qualification result.

Fig. 3 is reorganization plasmid enzyme restriction qualification result.

Fig. 4 is that the SDS-PAGE of expression product detects.

Embodiment:

The present invention will further specify through following examples, but the present invention is not limited.

Peanut Ara h2 gene order is analyzed, confirmed the RT-PCR primer, and it is synthetic to transfer to Shanghai living worker company.Take by weighing 2 gram peanut seeds, add liquid nitrogen grinding and become powder, adopt improved Trizol method to extract the total RNA of peanut.After obtaining total RNA, utilize ultraviolet spectrophotometer to measure it at 260nm, the ultraviolet light absorption value (A) at 280nm place, and the ratio of calculating A260/A280 are confirmed its purity.The RNA sample is through ultraviolet detection, and A260=1.159, productive rate are 211.93ug.g-1, and A260/ A280=1.90 shows that no protein or phenol pollute.

The RT-PCR reaction utilizes the RT-PCR cDNA first chain synthetic agent box of Roche company to synthesize first chain DNA, and the reverse transcription products therefrom is done template and carried out PCR reaction, pcr amplification condition: 94 ℃ of preparatory sex change 30s; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are continued to extend 10 min.PCR product Ago-Gel is carried out electrophoresis detection, adopt the plain edition agarose DNA of TIANGEN to reclaim kit purified pcr product, result such as accompanying drawing 1.

The PCR product that is recovered to is connected to the pET-32a carrier, and electrophoresis is identified.Prepare fresh Escherichia coli DH-5a competent cell with lime chloride, connect product transformed competence colibacillus cell.Bacterium original position PCR identifies that with the restriction endonuclease digestion demonstration transforms successfully result such as accompanying

drawing

2,3.

The abduction delivering of genes of interest, the single colony inoculation of transformant that contains recombinant plasmid in 3mL LB nutrient solution, 37 ℃ of thermal agitation overnight incubation, adding fresh LB nutrient solution and final concentration is the IPTG of 1mmol/L, continues culture bacteria liquid.Isolated cell, collecting precipitation carries out Protein Separation.With PBS damping fluid dissolving thalline, add protease inhibitors cocktail tablet, mixing.Use the ultrasonic disruption cell, the centrifuging and taking supernatant obtains the soluble protein extract.Through the existence of the visible destination protein of SDS-PAGE electrophoresis detection, shown in accompanying drawing 4.

Ara h 2 albumen of gained are concentrated, and antigen mixes with the equal-volume Freund through sterile purified water dilution back, and to make the final concentration of antigen be 0.2 mg/mL, and mixes even with turbine mixer.Get the subcutaneous multi-point injection of new zealand white rabbit and carry out immunity, initial immunity is used Freund's complete adjuvant; Booster immunization is used incomplete Freund.The serum that gather the immunity back tilts to place room temperature to place the 0.5h blood coagulation earlier, and 4 ℃ of refrigerators are placed and spent the night then, the packing of centrifuging serum, and-20 ℃ are frozen.Indirect ELISA is surveyed antibody titer, tire reach 1:105 after, be used for ELISA and detect.

Adopt the square formation titrimetry to confirm antigen and sero-fast optimum dilution degree.With the pure article of Ara h2, prepare the master sample of a series of variable concentrations, respectively with anti-hatching, join again and encapsulated in the certain density Ara h2 microwell plate; Through hatching, washing adds ELIAS secondary antibody, substrate; Stop, colour developing etc., ELIASA is measured light absorption value (OD).The A/A0 value of measuring with master sample is an ordinate, is horizontal ordinate with the master sample concentration C, sets up A/A0-log C typical curve.Shown in curve, thereby record the content of Arah2 in the sample.The competition of setting up thus based on standard antigen suppresses Arah2 content in the ELISA method detection sample to be tested.

Utilize the competition that standardization antigen sets up to suppress the ELISA method, detectability can reach below the 0.1mg/kg, and linear range is at sensing range 0.5～25mg/kg, sensitivity 0.25mg/kg.Adopt standardized antigen, error can reach 0.6% between the hole, and error can be controlled in 1.3% between plate, and it is high that the antigen-antibody of different batches is repeatedly measured repeatability, same testing sample batch between error below 2.1%.This shows that this method parameter in every respect all is much better than prior art, has splendid industrialization prospect.

Claims

1. ELISA detection method based on the peanut allergen protein Ara h2 of standard antigen; The dna sequence dna that comprises standard antigen obtains, the expression and purification of standard antigen, recombinant protein Polyclonal Antibody Preparation, suppress the ELISA system based on the competition of standard antigen and set up; It is characterized in that the accepted standard article are recombinant expressed Ara h2 albumen, and this albumen is used as required antibody in the antigen preparation ELISA detection.