CN102183658A - Recombinant UL55 protein-based duck plague virus antibody detection method - Google Patents

Abstract

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus (DPV) antibodies. The method specifically comprises the steps of preparation of solid phase antigens, primary antibody combination, secondary antibody combination, color development, detection, judgment and the like. The method is a purified recombinant UL55 protein-based indirect enzyme-linked immuno sorbent assay (ELISA) method, and has good specificity; the results of detecting the positive serums of duck virus hepatitis viruses (DHV), riemerella anatipestifer (RA), duck escherichia coli (E.coli), duck-derived salmonella, duck swollen head hemorrhagic disease viruses and duck-derived influenza viruses are negative; and the repeated tests in enzyme-labeled plates or between enzyme-labeled plates display that variation coefficients are less than 10 percent, and the method can detect out the positive serums of DPV weak virus vaccine immune ducks, wherein the DPV weak virus vaccine are diluted according to a ratio of 1:6400.

Description

A kind of duck plague virus antibody detection method based on reorganization UL55 albumen

Technical field

The present invention relates to the animal medicine field, particularly use reorganization UL55 prokaryotic expression protein and set up the duck plague virus antibody method of detection as envelope antigen.

Background technology

(duck plague DP) is a kind of height lethal infectious disease that is common in aquatic birds such as duck, goose, swan that is caused by the DPV in the herpetoviridae to duck plague.This sick mortality ratio height is propagated rapidly, from nineteen twenty-three after Holland takes place first, spread to world many countries and area, the development of foster duck industry in serious threat.Therefore press for and set up monitoring means effectively reliably, so that can in time correctly understand duck group's health status and immune antiboidy level.The classical diagnostic method of this disease is a neutralization test, but because of loaded down with trivial details (about 1～2 week) consuming time, susceptibility is relatively poor and requirement of experiment is strict, is unsuitable for the detection of blood serum sample in enormous quantities and promotes in basic unit.Detect the method that virus-specific antibodies has been acknowledged as a kind of sensitivity with the ELISA method, it is than neutralization test and hemagglutination-inhibition test sensitivity, and virus to being difficult for cultivating, or cultivation cycle long, be difficult for causing cytopathy, the virus that can't detect with neutralization test, ELISA detects and has more meaning, total antibody of certain virus can not only be detected, but also antibody can be detected at a certain antigenic component of virus.But, hindered bag by the large-scale application of the ELISA method (DPV-ELISA) of totivirus because the complicacy and the purity of DPV totivirus purification process are not ideal enough.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of is the duck plague virus antibody detection method of antigen based on reorganization UL55 albumen, the acquisition of described reorganization UL55 albumen is by making up prokaryotic expression plasmid pET32a-UL55, transforming and express bacterium and fermented and cultured, collect a large amount of bacterial sediments that contains reorganization UL55 albumen, obtain by ultrasonic disruption thalline, reorganization UL55 albumen inclusion body Xian Di, purifying and gradient dialysis again; Simultaneously, analyze this albumen of confirmation with Western blotting and can strong immune response take place with anti-DPV positive serum; The bag of further having determined reorganization UL55 albumen has been prepared the duck plague virus antibody detection method by concentration and one anti-, two anti-dilutabilitys, and this method has been avoided loaded down with trivial details viral purification step, and simple to operate, and specificity, sensitivity are higher.

For achieving the above object, technical scheme of the present invention is:

Based on the duck plague virus antibody detection method of reorganization UL55 albumen, concrete steps are:

The preparation of a solid phase antigen: will wrap by concentration and be that 0.2mg/ml concentration reorganization UL55 protein liquid solid phase carrier connects, with the confining liquid sealing, unconjugated antigen and impurity are removed in washing, solid phase antigen;

B one resistive connection closes: serum to be checked is done 160 times dilution, get dilute serum, promptly one is anti-, by insulation reaction described dilute serum combined with described solid phase antigen and form the solid phase antigen antibody complex, and impurity on the solid phase carrier is removed in washing;

C two resistive connections close: ELIAS secondary antibody is done 2000 times dilution, get the ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, get the anti-compound of Ag-Ab-two;

D colour developing: after the anti-compound of step c gained Ag-Ab-two adds the colour developing of chromogen substrate lucifuge, add the stop buffer cessation reaction sample liquid that must develop the color;

E detects and judges: the described colour developing sample liquid of steps d is surveyed OD with microplate reader _450nmValue, described OD _450nmValue＞0.330 is positive, described OD _450nm≤ 0.330 negative.

Further, the concrete steps of described duck plague virus antibody detection method are:

The preparation of a solid phase antigen: will wrap the reorganization UL55 protein liquid that is equaled 0.2mg/ml by concentration and be connected with solid phase carrier, with the confining liquid sealing, unconjugated antigen and impurity are removed in washing, solid phase antigen;

B one resistive connection closes: serum to be checked is done to equal 160 times dilution, get dilute serum, promptly one is anti-, by insulation reaction described dilute serum combined with described solid phase antigen and form the solid phase antigen antibody complex, and impurity on the solid phase carrier is removed in washing;

C two resistive connections close: ELIAS secondary antibody is done to equal 2000 times dilution, get the ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, get the anti-compound of Ag-Ab-two;

D colour developing: after the anti-compound of step c gained Ag-Ab-two adds the colour developing of chromogen substrate lucifuge, add the stop buffer cessation reaction sample liquid that must develop the color;

E detects and judges: the described colour developing sample liquid of steps d is surveyed OD with microplate reader _450nmValue, described OD _450nmValue＞0.330 is positive, described OD _450nm≤ 0.330 negative.

Further, solid phase carrier described in the step a is an ELISA Plate;

Further, the confining liquid described in the step a is that volumetric concentration is 1.0% bovine serum albumin(BSA);

Further, the time of lucifuge colour developing is 30 minutes among the step e;

Further, the addition of ELIAS secondary antibody dilution described in dilute serum and the step c described in the UL55 protein liquid of reorganization described in the step a, the step b is an equal-volume and more than or equal to 100 μ l;

Further, ELIAS secondary antibody described in the step c is goat-anti duck IgG-HRP;

Further, the substrate of chromogen described in the steps d is a tetramethyl benzidine;

Further, described method also comprises blank experiment and negative control experiment.

Beneficial effect of the present invention is: this method is based on the indirect elisa method of the reorganization UL55 albumen foundation of purifying, its specificity is good, positive serum to silent Salmonella (RA), duck Escherichia coli (E.coli), duck source salmonella (Salmonella), the swollen property haemorrhagic virus of duck and duck source influenza virus in duck virus hepatitis virus (DHV), the pest of duck detects, and the result is all negative; This method revision test in the ELISA Plate or between plate is shown that the Variation Lines number average less than 10%, can detect the positive serum through the DPV of 1: 6400 times of dilution attenuated vaccine immunity duck

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, and every all provide simultaneously original color picture and black and white that relates to protein expression shone.Relate to mark among the figure and implication is as follows:

Fig. 1-A is that the pcr amplification of DPV UL55 gene: M refers to Maker III relative molecular mass standard; The pcr amplification product that 1 finger is a template with normal DEF genomic DNA; 2 refer to DPV CHv strain genomic DNA to be the pcr amplification product (shown in the arrow is its molecular weight size, is about 799bp) of template; Fig. 1-B is that the double digestion of recombinant plasmid pMD18-UL55 is identified: M1 refers to relative molecular mass standard I II, and M2 is a DL2000 relative molecular mass standard; 1 refers to two fragments that recombinant plasmid pMD18-UL55 obtains with BamH I and Xho I double digestion; 2 refer to UL55PCR amplified production (the molecular weight size of small fragment shown in the arrow is about 799bp); Fig. 1-C is that single endonuclease digestion and the double digestion of recombinant expression carrier pET32a-UL55 identified: M1 is a DL2000 relative molecular mass standard; M2 refers to relative molecular mass standard DL15000; The 1st, the PCR product of UL55 gene; 2 refer to two fragments (the molecular weight size of small fragment shown in the arrow is about 799bp) that recombinant expression carrier pET32a-UL55 obtains with BamH I and Xho I double digestion; 3 refer to the linear plasmid that obtains after recombinant expression carrier pET32a-UL55 are with BamH I single endonuclease digestion.

Fig. 2-A is that the SDS-PAGE of recombinant expression protein identifies: M is a protein relative molecular mass standard; 1 for being that the pET32a zero load of expressive host adds IPTG and induces with BL21; 2, the 3 last cleer and peaceful inclusion bodys that are respectively the UL55 expression product that does not add derivant; 4,5 be respectively IPTG and induce the last cleer and peaceful inclusion body (the protein molecular weight size shown in the arrow is about 40KD) that is produced; Fig. 2-B is the different final concentration abduction delivering of derivant IPTG result: the IPTG concentration of 1-6 is respectively 1.0,0.8,, 0.6,0.4,0.2 and 0.1mmol/L; Fig. 2-C is that different temperatures induces the temperature of pET32a-UL55 expression of results: 1-3 to be respectively 37,30 and 25 ℃; Fig. 2-D is that different time induces the induction time of pET32a-UL55 expression of results: 1-5 to be respectively 4,5,6,7 and 8h.

Fig. 3 analyzes for reorganization UL55 protein purification effect and immunogenic detection: A, SDS-PAGE: M is protein standard (KD); 1 be behind the inclusion body washing method purifying again through the dialysis renaturation reorganization UL55 albumen; 2 is 5 reorganization UL55 inclusion body proteins after the washing; B, Western blotting analyze: the arrow indication is to be an anti-reorganization UL55 albumen (being about 20KD) that detects after the renaturation with the anti-DPV antibody of rabbit.

Embodiment

In order to make the purpose, technical solutions and advantages of the present invention clearer, the preferred embodiments of the present invention are described in detail below in conjunction with accompanying drawing.

The purifying of clone, prokaryotic expression and the UL55 albumen of embodiment 1 duck plague virus UL55 gene

1, material method

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

1.1 bacterial strain, plasmid and strain

Plasmid pMD18-T is available from the precious bioengineering in Dalian company limited; Prokaryotic expression plasmid pET32a (+), Novagen company product; Cloning host bacterium E.coli DH5a, expressive host bacterium E.coli BL21 (DE3) and DPV CHv velogen strain are provided by Sichuan Agricultural University poultry diease research centre.

1.2 test duck embryo and serum

10 age in days duck embryos, its kind duck DPV and antibody are all negative; The anti-DPV antibody of rabbit is provided by Sichuan Agricultural University poultry diease research centre.

1.3 main agents

Agarose, 2 * fool PCR Mixture, dna ligase Mixture, restriction enzyme BamH I and XhoI, UltraPureTM plasmid DNA are extracted kit and UltraPure in a small amount ^TMDNA reclaims kit equimolecular biological reagent and kit is matched Parkson gene technology company limited, Huamei Bio-Engrg Co., and Bio-Rad company available from the precious biotech firm in Dalian, Beijing; It is pure that other reagent is analysis, gives birth to worker Bioisystech Co., Ltd available from Shanghai.

2 experimental techniques

2.1 the clone of DPV UL55 gene

2.1.1 design of primers

Utilize Primer Premier5.0 software, with reference to UL55 gene order (GenBank accession number: EU071034), synthetic by precious biotinylated biomolecule technology company limited.Primer sequence:

F:5 '- GGATCCATGGCCGACGCGAAGGCGGT-3 ' (the line part is BamH I site);

R:5 '- CTCGAGGCTTGGGTCTTTACTTTTTGCGCGGAAC-3 ' (the line part is Xho I site).

After synthetic, with an amount of sterilization deionized water dissolving, making its final concentration is 20mmol/L, and-20 ℃ of preservations are standby.

2.1.2DPV the extraction of genomic DNA

2.1.2.1DEF method for making: get the healthy duck embryo of 10d age in days, use 5% tincture of iodine and 75% alcohol disinfecting eggshell surface respectively.Under the aseptic technique idiosome is taken out and idiosome is cleaned with PBS, cut off head, wing, leg and internal organ, after the PBS flushing idiosome is cut into the fritter of 1mm size, it is an amount of to add PBS, place in the triangular flask afterwards, add cell spreading agent (volume fraction is 2.5% trypsase) 150 μ l/ embryos, in 37 ℃ of water-baths, digest 3min.Immediately with cell suspension with the centrifugal 5min of 4000r/min, the tipping supernatant, after cell precipitation suspends with an amount of MEM, with 5 layers of filtered through gauze, in filtrate, add 10% calf serum and 100IU/ml two anti-after, be sub-packed in the 100ml Tissue Culture Flask, the 7ml/ bottle, level is statically placed in 37 ℃ of cell culture incubators and cultivates.

2.1.2.2 DPV propagation: get the DEF that just grows up to fine and close individual layer, abandon growth nutrient solution, behind sterilization PBS cleaning cell surface 2 times, adding DPV virus liquid 2～3ml covering cell surface adsorbs, abandon viral liquid behind 37 ℃ of absorption 120min, add then and contain the two anti-MEM of 3% calf serum and 100IU/ml and keep nutrient solution, 37 ℃ of cultivations afterwards.Do the DEF contrast that does not connect poison simultaneously.

2.1.2.3 DNA extraction method: the concrete steps of directly extracting the DPV genomic DNA from infection cell are as follows: (1) is chosen with DPV kind poison infected cell pathology (CPE) and is reached 60%～70% DEF (100ml cell bottle); Choosing the normal DEF of cellular morphology simultaneously compares; (2) cell culture fluid that inclines adds the cell pyrolysis liquid of 500 μ l, and adding Proteinase K (10mg/ml) to final concentration simultaneously is 200 μ g/ml, behind the mixing, hatches 10min for 37 ℃ gently; (3) cell suspending liquid is poured in the EP centrifuge tube, and remained in the interior lysate of cell bottle, pour in the centrifuge tube with the saturated phenol washing of 500 μ l; (4) with saturated phenol/chloroform and chloroform extracting 2 times, handle 2 times with the water saturation ether again; (5) add 1/10 times of volume 3mol/L NaAC, behind the mixing, add 2 times of cold absolute ethyl alcohols of volume, place 30～60min for-20 ℃; (6) the centrifugal 20min of 13000r/min, precipitation 70% ethanol washed twice of precooling; (7) after vacuum is drained, be dissolved in an amount of TE damping fluid, add 1 μ l RNA enzyme, 37 ℃ of effect 30min ,-20 ℃ of preservations are standby.

2.1.3PCR amplification DPV UL55 gene

The PCR reaction system is:

Mixing gently, the instantaneous centrifugal laggard performing PCR of 2000r/min.

Response parameter: 95 ℃ of pre-sex change 10min; 94 ℃ of sex change 40s, 66.2 ℃ of annealing 40s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min, standby in 4 ℃ of preservations.Get 4 μ l PCR products electrophoresis on 1% Ago-Gel, establish Maker III and blank, observe the length of amplified fragments.

2.1.4 the pMD18-T of UL55 gene clone, evaluation and order-checking

Optimizing under the good PCR condition with fidelity PCR Mixture amplification UL55 gene, product carries out the T clone and send the order-checking of the precious biotech firm in Dalian to confirm by the T clone kit instructions of the precious biotech firm in Dalian.

T is cloned bacterial classification inoculation in the LB of 5ml fluid nutrient medium (containing Amp 50 μ g/ml), 37 ℃ of water-bath jolting overnight incubation, extract recombinant plasmid next day, extracting method carries out the recombinant plasmid called after pMD18-UL55 of extracting by match Parkson, Beijing gene technology company limited plasmid extraction kit instructions.Then respectively with the digestion of BamH I/Xho I double digestion and the digestion of BamH I single endonuclease digestion, 1.0% gel electrophoresis observations.Do the pcr amplification genes of interest simultaneously.It is as follows that enzyme is cut system:

Enzyme is cut product carry out Preliminary Identification, and will clone product and send the evaluation of checking order of the precious biotech firm in Dalian with 1.0% agarose gel electrophoresis.

2.2 the structure of prokaryotic expression plasmid pET32a-UL55, abduction delivering and expression condition optimization

2.2.1 the structure of prokaryotic expression plasmid pET32a-UL55 and evaluation

2.2.1.1 the enzyme of purpose fragment is cut and is connected: restriction enzyme BamH I and Xho I be double digestion pMD18-UL55 plasmid and prokaryotic expression carrier pET32a (+) respectively, and the enzyme system of cutting is:

37 ℃ of water-bath 4h after reclaiming the kit operation instruction and reclaim the purpose fragment respectively by DNA, spend the night according to 16 ℃ of connections of following linked system.

2.2.1.2 the conversion of recombinant plasmid: adopt Calcium Chloride Method to prepare the DH5a competent cell.Afterwards, get connection liquid 10 μ l and be added in the centrifuge tube that contains 200 μ l competence DH5a ice bath 30min behind the mixing; Place 42 ℃ of water-bath 90sec, rapid then ice bath 2min; Add the LB fluid nutrient medium 800 μ l that do not contain Amp, 1～1.5h is cultivated in 37 ℃ of joltings (150r/min); Get 200 μ L cultures and coat the LB flat board that contains 100 μ g/ml Amp, 37 ℃ of overnight incubation, the single colony inoculation of picking next day is in the LB of 5ml fluid nutrient medium, cultivate 12～16h for 37 ℃, set up empty carrier conversion group (empty carrier 10 μ l+ competence DH5a 200 μ l), carrier-free control group (sterilization ultrapure water 10 μ l+ competence DH5a 200 μ l) simultaneously.

2.2.1.3 the enzyme of recombinant plasmid is cut with PCR and is identified: with clone's bacterial classification inoculation of above-mentioned preservation in the LB of 5ml fluid nutrient medium (containing Amp 50 μ g/ml), 37 ℃ of water-bath jolting overnight incubation, extract recombinant plasmid next day according to a conventional method, identify this recombinant plasmid with Xho I single endonuclease digestion, BamH I and Xho I double digestion then, it is as follows that its enzyme is cut system:

Then, be template with above-mentioned recombinant plasmid, utilize the primer among the method 2.1.1 to carry out the PCR reaction, its method and amplification condition are the same, get PCR product electrophoresis detection on 1.0% Ago-Gel.Cut evaluation through enzyme, obtain reorganization prokaryotic expression plasmid pET32a-UL55 with PCR.

2.2.2 the abduction delivering of recombinant expression plasmid pET32a-UL55

2.2.2.1 the extraction of recombinant plasmid pET32a-UL55: picking 2.2.1.3 has identified that the DH5a bacterial classification streak inoculation that contains positive recombinant plasmid pET32a-UL55 is on the LB agar plate that contains Amp 50g/ml, 37 ℃ of overnight incubation, get single colony inoculation next day on 5ml LB fluid nutrient medium, thermal agitation is cultivated 10～16h, centrifugal collection bacterium liquid is pressed UltraPure ^TMPlasmid DNA is extracted the extraction and purification of kit explanation carrying out recombinant plasmid in a small amount.

2.2.2.2 transforming, recombinant plasmid pET32a-UL55 expresses bacterium: adopt Calcium Chloride Method to prepare E.coli BL (DE3) competent cell, and the recombinant plasmid pET32a-UL55 of said extracted is transformed among the expressive host bacterium E.coli BL (DE3) the same 2.2.1.2 of method.

2.2.2.3 the abduction delivering of recombinant plasmid pET32a-UL55: from above-mentioned LB solid medium (containing Amp50 μ g/ml), picking positive colony bacterium, inoculation LB fluid nutrient medium, 37 ℃ of overnight incubation, get bacterium liquid next day in 1: 50 the ratio access 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml), thermal agitation is cultured to OD ₆₀₀=0.4 o'clock, add respectively IPTG to final concentration be 1.0mmol/L, after inducing 4h, collect 1ml and cultivate bacterium liquid, 4 ℃ of centrifugal 2min of 13000r/min abandon supernatant, add 80 μ l ultrapure waters and 20 μ l, 5 * SDS sample-loading buffer in the precipitation, 100 ℃ of water-bath heat denatured 5～10min carry out the 12%SDS-PAGE gel electrophoresis, observe expression of results.

2.2.2.4 the soluble analysis of recombinant plasmid pET32a-UL55 expression product: with the 100ml bacterium liquid of abduction delivering and the 100ml bacterium liquid of abduction delivering not, handle according to the following steps respectively: 4 ℃ of centrifugal 5min of 10000r/min, bacterial sediment suspends with 20ml 20mmol Tris-HCl (pH8.0); Put-20 ℃ spend the night after, adding lysozyme to final concentration is 1mg/ml, 4 ℃ are stirred 30min, ultrasound wave (ice bath) is broken thalline (600w, 30sec/ time, 10 times) intermittently, 4 ℃ of centrifugal 10min of 10000r/min, it is standby to get supernatant; Precipitation 10ml washing lotion (10mmol/L PBS+2mol/L urea+0.2%TritonX-100) suspend, 4 ℃, behind the centrifugal 10min of 10000r/min, precipitation suspends with the 10ml washing lotion once more, behind the repeated washing five times, with an amount of urea liquid (10mmol/L PBS+8mol/L urea) dissolution precipitation, cryopreservation is standby.Get the precipitation of an amount of last cleer and peaceful urea liquid dissolving respectively, to wherein adding 80 μ l ultrapure waters and 20 μ l, 5 * SDS sample-loading buffer, 100 ℃ of water-bath heat denatured 5～10min carry out the 12%SDS-PAGE gel electrophoresis, with gel with coomassie brilliant blue staining after, observations.

2.2.3 the optimization of recombinant plasmid pET32a-UL55 inductive condition

2.2.3.1 the concentration optimization of derivant IPTG: press the 2.2.2.3 method, get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-UL55, in the inoculation 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml), 37 ℃ of jolting overnight incubation.Changeed being inoculated in the 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) next day by 1: 50,37 ℃ of cultivations are cultured to OD ₆₀₀Be worth about 0.4 o'clock, get wherein 6 test tubes, add respectively after IPTG is 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 37 ℃ of inducing culture 4h of 0.8mmol/L, 1.0mmol/L to final concentration, as stated above sample is handled, the 12%SDS-PAGE electrophoresis, observations.

2.2.3.2 temperature conditions optimization: as stated above, get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-UL55, in the inoculation 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml), 37 ℃ of jolting overnight incubation.Changeed being inoculated in the 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) next day by 1: 50,37 ℃ of cultivations are cultured to OD ₆₀₀Be worth about 0.4 o'clock, and got wherein 3 test tubes, add respectively IPTG to final concentration be 0.2mmol/L, place 25 ℃, 30 ℃, 37 ℃ inducing culture 4h respectively, sample is handled 12%SDS-PAGE electrophoresis, observations by the 2.2.2.3 method.

2.2.3.3 induction time optimization: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET32a-UL55, on the inoculation 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml), 37 ℃ of jolting overnight incubation.Changeed being inoculated on the 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) next day by 1: 50, continue to be cultured to OD ₆₀₀Be worth about 0.4 o'clock, adding IPTG is 0.8mmol/L to final concentration, and 37 ℃ of inducing culture respectively at inducing back 4,5,6,7,8h, are drawn the 1ml nutrient solution, sample are handled 12%SDS-PAGE electrophoresis, observations by the 2.2.2.3 method.

2.3 a large amount of preparations, purifying and the renaturation of reorganization UL55 albumen

2.3.1 a large amount of cultivations of thalline

Concrete steps are: the expression bacterium E.coli BL21 (DE3) that (1) will contain the pET32a-UL55 plasmid is inoculated in the 200mlLB fluid nutrient medium (containing 50 μ g/ml Amp), cultivates 16h for 37 ℃, as kind of a daughter bacteria; (2) in sterilized LB fluid nutrient medium (containing 50 μ g/ml Amp), add kind of a daughter bacteria (ratio in 5%v/v adds); (3) at 180r/min, 37 ℃, cultivate under the condition of pH 7.0, wait to be cultured to bacterium liquid OD ₆₀₀During=0.6 left and right sides, adding IPTG is 0.2mmol/L to final concentration, 37 ℃ of inducing culture 4h; (4) collect cultured bacterium liquid (about 5L), collect bacterial sediment behind the centrifugal 10min of 8000r/min, with an amount of Tris-HCl (20mmol/L, pH 8.0) resuspended after, it is 1mg/ml that the adding lysozyme makes its final concentration ,-20 ℃ of preservations are standby.

2.3.2 the inclusion body washing method large-scale purification of reorganization UL55 albumen

Take out the bacterial sediment of-20 ℃ of preservations, after melting under the room temperature, ultrasound wave (ice bath) is broken thalline (200w, 30sec/ time, 5～10 times) intermittently, 4 ℃ of centrifugal 10min of 10000r/min.To precipitate with 20ml washing lotion (10mmol/LPBS+2mol/L urea+1%Triton X-100+20mM Tris-HCl+2mM EDTA) and suspend, behind 4 ℃ of centrifugal 10min of 10000r/min, precipitation suspends with the 20ml washing lotion once more, behind the repeated washing five times, with an amount of urea liquid (10mmol/LPBS+8M urea+50mM Tris-HCl+50mM NaCl+10% glycerine) dissolution precipitation, 4 ℃ of preservations are standby.

2.3.3 the renaturation and the detection of reorganization UL55 albumen

Inclusion body is washed the reorganization UL55 albumen that purifying obtains to join in the renaturation buffer (50mM Tris-HClpH 8.0+0.15M NaCl+1mM EDTA+1mM GSSG+10mM GSSH) several times, urea concentration is progressively reduced by 6M, 5M, 4M, 3M, 2M, make metaprotein renaturation gradually, and the control final concentration of protein is in the scope of 0.1～1mg/ml.4 ℃ of renaturation 24～48h, the protein liquid of collection dilution refolding, the 4 ℃ of dialysis of bag filter of packing into (dialysis buffer liquid: 24～48h 50mM Tris-HCl pH 8.0+50mM NaCl+0.5mM EDTA+10% glycerine+1% glycocoll).Dialysis back sample is collected supernatant behind the centrifugal 15min of 8000g, get wherein 20 μ l and carry out SDS-PAGE and analyze, and with purifying the anti-DPV of rabbit be one anti-, be that the two anti-Western blotting that carry out detect with HRP mark goat anti-rabbit igg.All the other protein liquids use the Bradford method to measure the ultimate density of albumen, and the packing postlyophilization concentrates standby.

Repeat twice of great expression and difference purifying UL55 recombinant protein again, obtain the UL55 recombinant protein of 3 batches of different expression and purifying altogether.

3 experimental results

3.1DPV the amplification of UL55 gene, T-clone and qualification result

3.1.1UL55 the pcr amplification result of gene

With DPV CHv strain genomic DNA is that template is carried out pcr amplification to the UL55 gene, its product is through 1.0% agarose gel electrophoresis, obtained the specific DNA band of a treaty 799bp, and be that template is carried out pcr amplification with normal DEF genomic DNA, no specific band, this is consistent with expected results (Fig. 1-A).

3.1.2UL55 gene T clones qualification result

The PCR product is connected and transformed competence colibacillus cell DH5 α with the pMD18-T carrier after glue reclaims purifying, the T that obtains clone called after pMD18-UL55.PMD18-UL55 is carried out PCR, enzyme cut that (Fig. 1-B) and order-checking identify that the result shows that the UL55 gene order that the T clone obtains is in full accord with known DPV UL55 gene order.

3.2 the structure of prokaryotic expression plasmid pET32a-UL55 and evaluation, abduction delivering and optimization result thereof

3.2.1 structure and the enzyme of recombinant expression plasmid pET32a-UL55 are cut evaluation

To reclaim the purpose segment behind BamH I and the Xho I double digestion T cloned plasmids, be connected with pET-32a (+) expression vector of cutting through same enzyme, transform DH5 α, obtain recombinant expression plasmid pET32a-UL55 (theoretical size is about 6169bp), the size of two segments that obtain behind BamH I and Xho I double digestion is about 5370bp and 799bp respectively and (sees Fig. 1-C), conform to theoretical value, show that prokaryotic expression carrier is successfully made up.

3.2.2 the abduction delivering of recombinant plasmid pET32a-UL55

3.2.2.1 the abduction delivering of recombinant plasmid pET32a-UL55: recombinant plasmid pET32a-UL55 is transformed expression strain BL21 (DE3) screened white colony containing on the LB agar plate of Amp.The expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET32a-UL55 with IPTG carry out abduction delivering, not with IPTG induce, empty carrier pET-32a (+) transforms the bacterial strain abduction delivering, the result shows: empty carrier pET-32a (+) transform bacterial strain inducing and not inducible strain the specific proteins band does not all appear; The reorganization UL55 albumen that recombinant expression plasmid pET32a-UL55 expresses is (Fig. 2-A) at the 40KD place.

3.2.2.2 the soluble analysis of recombinant plasmid pET32a-UL55 expression product: the 100ml bacterium liquid of abduction delivering is after soluble analysis is handled, electrophoresis result shows: expressing protein mainly is present in the precipitation, illustrates that recombinant expression protein exists with insoluble inclusion body form in thalline in a large number.

3.2.3 the optimization of recombinant plasmid pET32a-UL55 abduction delivering condition

3.2.3.1 the optimization of IPTG concentration: under 37 ℃ of conditions, add IPTG and make its final concentration be respectively 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L inducing culture 4h, the result shows: the control tube that does not add derivant does not have the specific proteins band; With increasing of IPTG concentration, protein induced amount does not have significant difference (Fig. 2-B).But when IPTG concentration was 0.2mmol/L, expressing quantity was slightly high comparatively speaking.Therefore, can select the IPTG concentration of 0.2mmol/L as abduction delivering concentration.

3.2.3.2 the optimization of inducing temperature condition: 37 ℃ of cultivations are cultured to OD ₆₀₀Be worth about 0.4 o'clock, get 3 sterilization test tubes, packing 5ml/ pipe, adding IPTG respectively is 0.2mmol/L to final concentration, and place 25 ℃, 30 ℃, 37 ℃ inducing culture 4h respectively, the result: temperature is in the time of 25 ℃, the inducible protein amount is less, (Fig. 2-C), illustrate that protein induced amount increases gradually along with temperature raises the highest in the time of 37 ℃.Therefore, selecting temperature is best inducing temperature for 37 ℃.

3.2.3.3 the optimization of induction time: in IPTG concentration is 0.2mmol/L, under 37 ℃ of conditions, adopts the different induction times of 4～8h to carry out abduction delivering, and the result induces the recombinant protein table amount reduction of generation along with the increase of time.Induce the expression of recombinant proteins amount of 5～8h all significantly to be lower than 4h and induce group; And induce 6～8h, its expression of recombinant proteins amount to be lower than 5h and compare no significant change (Fig. 2-D) to each other.Therefore, select 4h as best induction time.

3.3 the purification result of reorganization UL55 albumen

By a large amount of enlarged culture, collected the bacterial sediment that contains reorganization UL55 albumen in a large number, through the lysozyme cracking, ultrasonication, washing and dissolving inclusion body, processes such as metaprotein dialysis renaturation have obtained the reorganization UL55 albumen of large-scale purification, analyze to show that by SDS-PAGE the reorganization UL55 albumen of purifying has higher purity (Fig. 3-A), Western blotting analyze to show that this reorganization UL55 albumen can strong immune response (Fig. 3-B) take place with anti-DPV positive serum, show that this recombinant protein has higher immunogenicity, can be used as and detect duck plague detection of antibodies antigen clinically.

Embodiment 2, UL55-ELISA method detect the foundation and the application of DPV antibody method

The reorganization UL55 albumen that the foregoing description obtains, anti-DPV duck serum (is the immune duck serum of 14d behind the attenuated vaccine immunity, it is 1: 8 that neutralization is tired), anti-DHV (DHV), RA (Mo Shi bacillus in the duck), Salmonella (salmonella), duck swollen a haemorrhagic virus, influenza virus and E.coli (Escherichia coli) duck serum and non-immune duck negative serum are provided by Sichuan Agricultural University poultry diease research centre; Goat-anti duck IgG-HRP (the goat-anti duck IgG of horseradish peroxidase mark) and tetramethyl benzidine (TMB) are all available from U.S. KPL company; Bovine serum albumin(BSA) (BSA) is available from U.S. Sigma company product.

The 1UL55-ELISA method detects the foundation of DPV antibody method

1.1 reorganization UL55 albumen bag determining by concentration and serum dilution

Adopt the square formation method to determine best antigen coated concentration and serum dilute concentration, be that reorganization UL55 albumen after the renaturation of 4mg/ml carried out serial dilution (1: 10 with coating buffer to concentration, 1: 20,1: 40,1: 80,1: 160,1: 320), bag is by enzyme reaction plate, every hole bag is by 100 μ l, the parallel bag of each titre is by two row, duck serum (anti-DPV positive serum or non-immune duck negative serum) was pressed 1: 10,1: 20,1: 40,1: 80,1: 160,1: 320 dilutability carries out serial dilution, goat-anti duck IgG-HRP does dilution in 1: 2000, measure with indirect ELISA method, select the OD of positive serum _450nmValue is near 1.0, the OD of negative serum _450nmBe worth lessly, the UL55 albumen that the P/N value is maximum and the dilutability of duck serum be the suitableeest working concentration of the antigen-antibody reaction of ELISA for this reason.

After reorganization UL55 proteantigen is made serial dilution with coating buffer, add 1 again: 10-1: the positive serum or the negative serum of 320 dilutions; Other adds the goat-anti duck IgG-HRP ELIAS secondary antibody of dilution in 1: 2000, adds substrate colour developing liquid, measures OD with full-automatic microplate reader _450nmValue.Between 0.4mg/ml～0.0125mg/ml the time, the dilutability of positive serum is 1 to the purifying antigen bag: 10-1 by concentration: 320 o'clock, and the OD of different dilution antibody _450nmValue sees Table 1.When the P/N value was 9.667 to the maximum, when being 0.2mg/ml (the antigen diluent degree is 1: 20) by the best antigen coated concentration of square formation titration gained, the dilutability of positive serum was 1: 160.The P/N value of each antigen-antibody concentration correspondence sees Table 2.

The OD of table 1 square formation titration _450nmThe result

The best bag of table 2 DPV-UL55 expressing protein determining by concentration and serum-concentration (P/N value)

1.2 determining of ELIAS secondary antibody optimum dilution degree

The concentration bag that the reorganization UL55 proteantigen of purifying is pressed 0.2mg/ml is by in the ELISA reaction plate, and every hole 100 μ l add the positive serum or the negative serum of dilution in 1: 160 again.Afterwards enzyme labelled antibody is done and caught ELISA mensuration in 1: 500,1: 1000,1: 2000,1: 4000 behind the serial dilution, detect the normal cell culture simultaneously and substitute the OD that duck plague virus makes negative control _450nmValue, enzyme labelled antibody concentration is as the suitableeest working concentration when selecting the P/N value maximum.Dilution in the present embodiment is all diluted according to volume ratio.

ELISA Plate is the reorganization UL55 proteantigen bag quilt of 0.2mg/ml with bag by concentration, and serum is done dilution in 1: 160, and enzyme labelled antibody is done serial dilution, carries out the indirect ELISA detection, and the result is that P/N value under each serum to be checked and the ELIAS secondary antibody concentration is all widely greater than 2.1.When the dilutability of ELIAS secondary antibody at 1: 2000, detect serum-concentration 1: 160 o'clock, P/N value maximum; Therefore the best two anti-dilutabilitys of selecting are 1: 2000.

1.3UL55-ELISA the trace routine of determining to reach the UL55-ELISA method of yin and yang attribute critical value

Get 30 parts of non-immune duck negative serums, the parallel bag of each sample is by 1 time.Reorganization UL55 albumen bag is carried out indirect ELISA by concentration, serum to be checked and ELIAS secondary antibody optimum dilution degree measure, to determine duck serum its absorption value scope when no DPV infects.Simultaneously make blank with 1.0%BSA/PBS solution.OD with 30 parts of blood serum samples _450nmThe mean value (X) of value and 3 times of standard variances (SD) sum are as the upper limit of negative serum, the i.e. OD of blood serum sample to be checked _450nmDuring value＞X+3SD, be judged as the positive; Otherwise it is negative.

By 30 parts of DPV negative serums are detected (table 3), the result shows that healthy duck serum OD value is up to 0.347, and the X value is 0.148, and the SD value is 0.061, and critical value (X+3SD) is 0.330.The OD value that is testing sample is positive greater than 0.330, be less than or equal to 0.330 negative.

20 parts of DPV negative serums of table 3 testing result

1.4UL55-ELISA the trace routine of method

According to above-mentioned optimization result, the trace routine of indirect ELISA method is as follows: the preparation of (1) solid phase antigen: the UL55 albumen of will recombinating with 0.2mg/ml concentration bag by in ELISA Plate, 100 μ l/ holes, put 4 ℃ of wet box overnight incubation, wash the plate machine washing and wash 3 times, 5min/ time, pat dry; Add 100 μ l/ hole confining liquids, 37 ℃ of wet box sealing 1h wash the plate machine washing and wash 1 time, 5min/ time, pat dry; (2) one resistive connections close: with the volume dilution degree dilution back adding ELISA Plate of serum to be checked by 1: 160, and 100 μ l/L, 37 ℃ of wet boxes are hatched 1h, wash the plate machine washing and wash 3 times, 5min/ time, pat dry; (3) two resistive connections close: with the ELIAS secondary antibody dilution volume dilution degree dilution back of ELIAS secondary antibody (goat-anti duck IgG-HRP) with 1: 2000 added, and 100 μ l/ holes, 37 ℃ of reaction 45min wash the plate machine washing and wash 3 times, 5min/ time, pat dry; (4) colour developing: add tmb substrate (chromogen substrate tetramethyl benzidine) 100 μ l/ holes, behind 25 ℃ of lucifuge colour developing 30min, add 50 μ l/ hole 2mol/L sulfuric acid cessation reactions; (5) detection and judgement: survey OD with microplate reader _450nmValue is when microplate reader is surveyed OD _450nmValue, OD _450nmValue is positive greater than 0.330, be less than or equal to 0.330 negative.Simultaneously make parallel blank with 1.0% BSA/PBS solution.

2 beneficial effect evaluations

2.1 susceptibility experiment

The UL55 albumen of will recombinating is wrapped quilt by the best bag by concentration, it is that 1: 8 positive serum was done 1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800,1: 25600 that 14d behind the DPV attenuated vaccine immunity, neutralization are tired, 1: 51200 totally 9 volume dilution degree, all the other conditions are carried out the UL55-ELISA test by the present embodiment condition, and the result is as shown in table 4.When positive serum is diluted to 1: 6400, be difficult to judge negative, positive findings by the visual inspection change color, but still can detect by microplate reader, positive serum testing result subcritical value 0.330 by dilution in 1: 12800, show that the method can detect the positive serum of 14d behind the attenuated vaccine immunity of 1: 6400 times of dilution, has stronger susceptibility.

Table 4 sensitivity tests testing result

P is positive, and N is negative;

2.2 specificity test findings

Adopt the ELISA condition of establishing, with the UL55 albumen bag of purifying by the ELISA ELISA Plate, carry out the test of UL55-ELISA specificity for each two parts with DPV positive serum, DHV positive serum, RA positive serum, E.coli positive serum, Salmonella positive serum, a swollen property haemorrhage positive serum, influenza positive serum, the result is as shown in table 5.According to critical value criterion, 2 parts of DPV positive serum OD _450nmBe 0.925,0.896,, be judged to the positive, all the other each virosis or bacterial diseases, duck serum OD all much larger than 0.330 _450nmValue is all less than critical value, and is then all negative, shows that the UL55-ELISA method of the detection DPV antibody of foundation has excellent specificity.

Table 5 specificity test findings

P is positive, and N is negative;

2.3 barrier test

The DPV positive serum is pressed the optimum dilution degree dilution, add the expressing protein UL55 of equivalent optimum dilution degree, behind 37 ℃ of effect 1h, carry out indirect ELISA by the ELISA condition of establishing, establish control group, comparative result simultaneously with the DPV positive serum as an antiserum.Calculate (N-P)/N=(OD450nm value before the blocking-up-blocking-up back OD450nm value)/OD450nm value before blocking, if this value greater than 0.5, then is judged to the blocking-up positive, otherwise negative.The result shows, the positive serum OD450nm value of duck plague virus UL55 proteantigen blocking-up is obviously lower than unbroken contrast positive serum, block back three parts of positive OD450nm values and on average be respectively 0.111,0.120,0.100, the 0D450nm value that positive serum is not blocked in contrast on average is respectively 0.708,0.675,0.637.This moment, (N-P)/N value of expressing protein also was respectively 0.84,0.82,0.84, all greater than 0.5, was judged to the blocking-up positive, illustrated that expressing protein UL24 antigen can block positive serum, the results are shown in Table 6.

Table 6 barrier test findings

3, discuss

In ELISA method testing process, antigen coated concentration is bigger to the test findings influence, if antigen coated concentration height, owing to the big multiple stratification (stacking efect) that causes protein molecular of acting force between the antigen protein molecule, be washed non-specific increase easily, if concentration is too low, the ELISA Plate surface may stay not adsorption antigen but the active surface of complete closed, non-specificly also can increase, therefore must screening the coating protein antigen concentration.In addition; the purity of antibody is directly connected to the specificity and the susceptibility of ELISA test; the antibody that purity is not high usually has the host cell macromolecular compound that combines with specific antigen of excess; can be with the limited carrier surface position of antigenic competition and reduce effective adsorption rate; therefore highly purified antibody protein can improve atopic; but indirect ELISA method detects the antibody in the serum usually; the serum composition complexity; usually one by one tested serum is not carried out purifying in the production; have only by groping suitable serum diluting multiple, could reduce non-specific bond.This experiment square formation method is determined the optimum diluting multiple of serum (one anti-), from the result, though serum diluting multiple from 1: 10 to 1: 320, serum-concentration reduces, OD _450nmValue diminishes, according to OD _450nmValue is near 1.0, and the P/N maximum is judged the optimum dilution degree of antigen-antibody.Therefore the best antigen coated concentration of determining is 0.2mg/ml, and the extension rate of serum is 1: 160.

The height of ELIAS secondary antibody concentration also has bigger influence to test findings, and concentration is too high, and the chance of non-specific binding increases, and false positive may occur, and concentration is too low, does not have effective combination, phenomenons such as false negative then may occur.This experiment is carried out the optimum diluting multiple that indirect ELISA is determined ELIAS secondary antibody under the good ELISA condition of optimization.According to OD _450nmValue is near 1.0, and the P/N maximum is judged optimum dilution degree, and the optimum diluting multiple of determining ELIAS secondary antibody is 1: 2000.Sealing is the process of wrapping quilt again with the irrelevant protein solution of high concentration after the bag quilt, concentration used when antigen coated is lower, absorb the back surface of solid phase carriers space that is not occupied is still arranged, sealing is exactly to allow these spaces of a large amount of incoherent protein fillings, thereby repels the absorption again of interfering material in ELISA step thereafter.The most frequently used sealer is the 0.5%-1.0% bovine serum albumin(BSA), and also useful skimmed milk power or 1.0% gelatin are as sealer.Skimmed milk power also is a kind of good sealer, and its maximum characteristics are inexpensive, but because the composition complexity of milk powder, and the carrier after the sealing is difficult for long preservation, therefore less application in the preparation of kit.Select for use 1.0% BSA to make confining liquid in the present embodiment, its composition is single, and sealing effect is better.

In ELISA criterion, two kinds of methods commonly used, a kind of is with the original OD that measures sample _450nmValue and standard variance are determined critical value.Another kind is to calculate the ratio of testing sample with respect to negative control, and the frequency of tiring by the mensuration great amount of samples distributes to determine critical value and suspicious interval.This research has been adopted first kind of criterion when definite yin and yang attribute criterion.OD according to a large amount of negative samples _450nmValue adopts statistical method to determine the negative and positive critical value.The principle of foundation is the OD of sample _450nmValue＞negative sample OD _450nmDuring the mean value (X)+3SD of value, can on 99.9% level, be judged to the positive, according to principle of statistics, a large amount of experiment confirms this criterion be reliable.In addition, when reports such as Alonso utilize the albumen of Bacillus coli expression to detect the field porcine blood serum as envelope antigen, the problem that may exist host protein to disturb, the Escherichia coli antibody that promptly exists in the porcine blood serum might with remaining host protein generation nonspecific reaction in the expressing protein.Therefore, in the process of setting up the UL55-ELISA method, selected the E.coli positive serum in duck source to do the specificity experiment, the result shows this UL55-ELISA method and E.coli serum no cross reaction, and specificity is good.Do the specificity test with DPV positive serum, DHV positive serum, RA positive serum, E.coli positive serum, Salmonella positive serum, a swollen property haemorrhage positive serum, influenza positive serum again simultaneously, the result has confirmed that the UL55-ELISA that sets up has good specificity and susceptibility, shown good prospects for application, also laid a good foundation for further being assembled into kit.

Embodiment 3

Another example, the trace routine of indirect ELISA method is as follows: the preparation of (1) solid phase antigen: by in ELISA Plate, 4 ℃ of wet box overnight incubation are put in 100 μ l/ holes to the UL55 albumen of will recombinating with 0.2mg/ml concentration bag, wash the plate machine washing and wash 3 times, 5min/ time, pat dry; Add 100 μ l/ hole confining liquids, 37 ℃ of wet box sealing 1h wash the plate machine washing and wash 1 time, 5min/ time, pat dry; (2) one resistive connections close: serum to be checked is added ELISA Plate by 1: 20 volume dilution degree dilution back, 100 μ l/L, 37 ℃ of wet boxes are hatched 1h, wash the plate machine washing and wash 3 times, 5min/ time, clap in; (3) two resistive connections close: with the ELIAS secondary antibody dilution volume dilution degree dilution back of ELIAS secondary antibody (goat-anti duck IgG-HRP) with 1: 1000 added, and 100 μ l/ holes, 37 ℃ of reaction 45min wash the plate machine washing and wash 3 times, 5min/ time, pat dry; (4) colour developing: add tmb substrate (chromogen substrate tetramethyl benzidine) 100 μ l/ holes, behind 25 ℃ of lucifuge colour developing 30min, add 50 μ l/ hole 2mol/L sulfuric acid cessation reactions; (5) detection and judgement: survey OD with microplate reader _450nmValue is when microplate reader is surveyed OD _450nmValue, OD _450nmValue is positive greater than 0.330, be less than or equal to 0.330 negative.Simultaneously make parallel blank with 1.0% BSA/PBS solution.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Claims (8)

1. based on the duck plague virus antibody detection method of reorganization UL55 albumen, it is characterized in that concrete steps are:

The preparation of a solid phase antigen: will wrap by concentration is the reorganization UL55 protein liquid solid phase carrier connection of 0.2mg/ml, and with the confining liquid sealing, unconjugated antigen and impurity are removed in washing, get solid phase antigen;

B one resistive connection closes: serum to be checked is done 160 times dilution, get dilute serum, promptly one is anti-, by insulation reaction described dilute serum combined with described solid phase antigen and form the solid phase antigen antibody complex, and impurity on the solid phase carrier is removed in washing;

C two resistive connections close: ELIAS secondary antibody is done 2000 times dilution, get the ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, get the anti-compound of Ag-Ab-two;

D colour developing: after the anti-compound of step c gained Ag-Ab-two adds the colour developing of chromogen substrate lucifuge, add the stop buffer cessation reaction sample liquid that must develop the color;

E detects and judges: the described colour developing sample liquid of steps d is surveyed OD with microplate reader _450nmValue, described OD _450nmValue＞0.330 is positive, described OD _450nm≤ 0.330 negative.

2. duck plague virus antibody detection method according to claim 1 is characterized in that: solid phase carrier described in the step a is an ELISA Plate.

3. duck plague virus antibody detection method according to claim 1 is characterized in that: the confining liquid described in the step a is that volumetric concentration is 1.0% bovine serum albumin(BSA).

4. duck plague virus antibody detection method according to claim 1 is characterized in that: the time of lucifuge colour developing is 30 minutes in the steps d.

5. duck plague virus antibody detection method according to claim 1 is characterized in that: the addition of ELIAS secondary antibody dilution described in dilute serum and the step c described in the UL55 protein liquid of reorganization described in the step a, the step b is an equal-volume and more than or equal to 100 μ l.

6. the method that is used to detect duck plague virus antibody according to claim 1 is characterized in that: ELIAS secondary antibody described in the step c is goat-anti duck IgG-HRP.

7. duck plague virus antibody detection method according to claim 1 is characterized in that: the substrate of chromogen described in the steps d is a tetramethyl benzidine.

8. duck plague virus antibody detection method according to claim 1 is characterized in that: described method also comprises blank experiment and negative control experiment.

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