CN102183658B

CN102183658B - Recombinant UL55 protein-based duck plague virus antibody detection method

Info

Publication number: CN102183658B
Application number: CN201110047062.4A
Authority: CN
Inventors: 程安春; 吴英; 汪铭书; 陈孝跃
Original assignee: ENGINEERING AND TECHONLOGY CENTER FOR LABORATORY ANIMALS OF SICHUAN AGRICULTURAL UNIVERSITY
Current assignee: Sichuan Agricultural University
Priority date: 2011-02-28
Filing date: 2011-02-28
Publication date: 2014-03-19
Anticipated expiration: 2031-02-28
Also published as: CN102183658A

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 restructuring UL55 albumen

Technical field

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

Background technology

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

Summary of the invention

In view of this, the object of the present invention is to provide a kind of duck plague virus antibody detection method that is antigen based on restructuring UL55 albumen, the acquisition of described restructuring UL55 albumen is by building prokaryotic expression plasmid pET32a-UL55, transforming and express bacterium fermented and cultured, collect a large amount of bacterial sediments that contains restructuring UL55 albumen, then obtain by ultrasonic disruption thalline, restructuring UL55 albumen inclusion body Xian Di, purifying and gradient dialysis; Meanwhile, with Western blotting, analyze and confirm that this albumen can strong immune response occur with anti-DPV positive serum; Further determined the coated concentration of restructuring UL55 albumen and primary antibodie, two anti-dilutabilitys, prepared duck plague virus antibody detection method, the 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:

Duck plague virus antibody detection method based on restructuring UL55 albumen, concrete steps are:

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

B mono-anti-binding: serum to be checked is done to the dilution of 160 times, obtained dilute serum, primary antibodie, makes described dilute serum be combined with described solid phase antigen by insulation reaction and form solid phase antigen antibody complex, and impurity on solid phase carrier is removed in washing;

C bis-anti-bindings: ELIAS secondary antibody is done to the dilution of 2000 times, obtained ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, obtain the anti-compound of Ag-Ab-bis-;

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

E detects and judges: by developing the color described in steps d, sample liquid is surveyed OD by microplate reader _450nmvalue, described OD _450nm> 0.330 is positive for value, 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: the restructuring UL55 protein liquid that coated concentration is equaled to 0.2mg/ml is connected with solid phase carrier, with confining liquid sealing, unconjugated antigen and impurity are removed in washing, obtain solid phase antigen;

B mono-anti-binding: serum to be checked is done to equal the dilution of 160 times, obtain dilute serum, primary antibodie, makes described dilute serum be combined with described solid phase antigen by insulation reaction and form solid phase antigen antibody complex, and impurity on solid phase carrier is removed in washing;

C bis-anti-bindings: ELIAS secondary antibody is done to equal the dilution of 2000 times, obtain ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, obtain the anti-compound of Ag-Ab-bis-;

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

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

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

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

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

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

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

Beneficial effect of the present invention is: this method is the indirect elisa method that the restructuring UL55 albumen based on purifying is set up, its specificity is good, positive serum to duck virus hepatitis virus (DHV), Riemerellosis Anatipestifer (RA), E. coli isolated from ducks (E.coli), duck source salmonella (Salmonella), the swollen property haemorrhagic virus of duck and duck source and course Influenza Virus detects, and result is all negative; The method revision test in ELISA Plate or between plate is shown to the coefficient of variation is all less than 10%, can detect the positive serum through the DPV of 1: 6400 times of dilution attenuated vaccine immunity duck

Accompanying drawing explanation

In order to make the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the present invention is described in further detail, and every all simultaneously provide original color picture and black and white that relates to protein expression shone.In figure, relate to mark 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; 1 finger be take the pcr amplification product that normal DEF genomic DNA is template; 2 fingers be take the pcr amplification product (shown in arrow is its molecular size range, is about 799bp) that DPV CHv strain genomic DNA is 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 DL2000 relative molecular mass standard; Two fragments that 1 finger recombinant plasmid pMD18-UL55 obtains with BamH I and Xho I double digestion; 2 finger UL55PCR amplified productions (molecular size range of small fragment shown in 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 DL2000 relative molecular mass standard; M2 refers to relative molecular mass standard DL15000; The PCR product of the 1st, UL55 gene; Two fragments (molecular size range of small fragment shown in arrow is about 799bp) that 2 finger recombinant expression carrier pET32a-UL55 obtain with BamH I and Xho I double digestion; 3 linear plasmids that obtain after BamH I single endonuclease digestion for finger recombinant expression carrier pET32a-UL55.

Fig. 2-A is that the SDS-PAGE of recombinant expression protein identifies: M is protein relative molecular mass standard; 1 adds IPTG induction for take the pET32a zero load that BL21 is expressive host; 2, the 3 upper cleer and peaceful inclusion bodys that are respectively the UL55 expression product that does not add derivant; 4,5 be respectively the upper cleer and peaceful inclusion body (the protein molecular weight size shown in arrow is about 40KD) that IPTG induction produces; 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 different temperatures induction pET32a-UL55 expression of results: the temperature of 1-3 is respectively 37,30 and 25 ℃; Fig. 2-D is different time induction pET32a-UL55 expression of results: the induction time of 1-5 is respectively 4,5,6,7 and 8h.

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

Embodiment

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

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 preferred embodiment, conventionally according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

1.1 bacterial strains, plasmid and strain

Plasmid pMD18-T, purchased from Dalian precious bioengineering 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, provided by Sichuan Agricultural University's poultry diease research centre.

1.2 test duck embryo and serum

10 age in days duck embryos, its kind of duck DPV and antibody are all negative; The anti-DPV antibody of rabbit, is provided by Sichuan Agricultural University's 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 recovery kit equimolecular biological reagent and kit are purchased from the precious biotech firm in Dalian, match Parkson, Beijing gene technology company limited, Huamei Bio-Engrg Co., and Bio-Rad company; It is pure that other reagent is analysis, purchased from Shanghai Sheng Gong Bioisystech Co., Ltd.

2 experimental techniques

The clone of 2.1 DPV UL55 genes

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 ' (line part is BamH I site);

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

After synthetic, with appropriate sterilizing deionized water dissolving, making its final concentration is 20mmol/L, and-20 ℃ save backup.

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 respectively 5% tincture of iodine and 75% alcohol disinfecting eggshell surface.Under aseptic technique, idiosome taken out and with PBS, idiosome cleaned, cut off head, wing, leg and internal organ, after PBS rinses, idiosome is cut into the fritter of 1mm size, add PBS appropriate, be placed in afterwards in triangular flask, add cell-dispersing agent (trypsase that volume fraction is 2.5%) 150 μ l/ embryos, in 37 ℃ of water-baths, digest 3min.Immediately by cell suspension with the centrifugal 5min of 4000r/min, tipping supernatant, after cell precipitation suspends with appropriate MEM, by 5 layers of filtered through gauze, after adding in filtrate 10% calf serum and 100IU/ml dual anti-, be sub-packed in 100ml Tissue Culture Flask, 7ml/ bottle, level is statically placed in 37 ℃ of cell culture incubators and cultivates.

2.1.2.2 DPV breeds: get the DEF that just grows up to fine and close individual layer, abandon growth nutrient solution, with after sterilizing PBS cleaning cell surface 2 times, adding DPV virus liquid 2～3ml to cover cell surface adsorbs, after 37 ℃ of absorption 120min, abandon virus liquid, then add containing 3% calf serum and the dual anti-MEM of 100IU/ml and maintain nutrient solution, 37 ℃ of cultivations afterwards.Do simultaneously and do not connect malicious DEF contrast.

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

2.1.3PCR DPV UL55 gene increases

PCR reaction system is:

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

Response parameter: 95 ℃ of denaturation 10min; 94 ℃ of sex change 40s, 66.2 ℃ of annealing 40s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min, in 4 ℃, save backup.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

Under the PCR condition of having optimized, with fidelity PCR Mixture amplification UL55 gene, product carries out 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 to bacterial classification to be inoculated in the LB fluid nutrient medium (containing Amp 50 μ g/ml) of 5ml, 37 ℃ of water-bath jolting overnight incubation, extract recombinant plasmid next day, extracting method is undertaken by match Parkson, Beijing gene technology company limited plasmid extraction kit instructions, the recombinant plasmid called after pMD18-UL55 of extracting.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 pcr amplification genes of interest simultaneously.It is as follows that enzyme is cut system:

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

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

2.2.1 the Construction and identification of prokaryotic expression plasmid pET32a-UL55

2.2.1.1 the enzyme of object 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, reclaim kit operation instruction by DNA and reclaim respectively after object fragment, according to 16 ℃ of connections of following linked system, spend the night.

2.2.1.2 the conversion of recombinant plasmid: adopt Calcium Chloride Method to prepare DH5a competent cell.Afterwards, get connecting fluid 10 μ l and be added to containing in the centrifuge tube of 200 μ l competence DH5a, mix rear ice bath 30min; Be placed in 42 ℃ of water-bath 90sec, then rapid ice bath 2min; Add not containing the LB fluid nutrient medium 800 μ l of Amp, 1～1.5h is cultivated in 37 ℃ of joltings (150r/min); Getting 200 μ L cultures coats containing the LB of 100 μ g/ml Amp dull and stereotyped, 37 ℃ of overnight incubation, the single colony inoculation of picking next day is in the LB of 5ml fluid nutrient medium, cultivate 12～16h, set up empty carrier conversion group (empty carrier 10 μ l+ competence DH5a 200 μ l), carrier-free control group (sterilizing ultrapure water 10 μ l+ competence DH5a 200 μ l) simultaneously for 37 ℃.

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

Then, take above-mentioned recombinant plasmid as template, utilize the primer in method 2.1.1 to carry out PCR reaction, its method and amplification condition are the same, get PCR product electrophoresis detection on 1.0% Ago-Gel.Through enzyme, cut with PCR and identify, obtain recombined pronucleus expression plasmid pET32a-UL55.

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 in containing on the LB agar plate of 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, by UltraPure ^tMplasmid DNA is extracted the extraction and purification that recombinant plasmid is carried out in kit explanation in a small amount.

2.2.2.2 recombinant plasmid pET32a-UL55 transforms and 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 in expressive host bacterium E.coli BL (DE3) to 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 the ratio access 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) of 1: 50, thermal agitation is cultured to OD ₆₀₀=0.4 o'clock, add respectively IPTG to final concentration be 1.0mmol/L, after induction 4h, collect 1ml and cultivate bacterium liquid, 4 ℃ of centrifugal 2min of 13000r/min, abandon supernatant, in precipitation, add 80 μ l ultrapure waters and 20 μ l 5 * SDS sample-loading buffers, 100 ℃ of heating water bath sex change 5～10min, carry out 12%SDS-PAGE gel electrophoresis, observe expression of results.

2.2.2.4 the soluble analysis of recombinant plasmid pET32a-UL55 expression product: by the 100ml bacterium liquid of abduction delivering and the 100ml bacterium liquid of abduction delivering not, process 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, get supernatant standby; Precipitation suspends by 10ml washing lotion (10mmol/L PBS+2mol/L urea+0.2%TritonX-100), 4 ℃, after the centrifugal 10min of 10000r/min, precipitation suspends by 10ml washing lotion again, after repeated washing five times, with appropriate urea liquid (10mmol/L PBS+8mol/L urea) dissolution precipitation, low temperature saves backup.Get respectively the precipitation that appropriate upper cleer and peaceful urea liquid dissolves, add wherein 80 μ l ultrapure waters and 20 μ l 5 * SDS sample-loading buffers, 100 ℃ of heating water bath sex change 5～10min, carry out 12%SDS-PAGE gel electrophoresis, by gel with after coomassie brilliant blue staining, observations.

2.2.3 the optimization of recombinant plasmid pET32a-UL55 inductive condition

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

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

2.2.3.3 induction time optimization: get the expressive host bacterium BL21 (DE3) containing recombinant plasmid pET32a-UL55, inoculation 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) is upper, 37 ℃ of jolting overnight incubation.Turned and be inoculated on 5ml LB fluid nutrient medium (containing Amp 50 μ g/ml) next day by 1: 50, continues to be cultured to OD ₆₀₀be worth approximately 0.4 o'clock, adding IPTG is 0.8mmol/L to final concentration, and 37 ℃ of inductions are cultivated, respectively at after

induction

4,5,6,7,8h, draw 1ml nutrient solution, by 2.2.2.3 method, sample is processed 12%SDS-PAGE electrophoresis, observations.

A large amount of preparations, purifying and the renaturation of 2.3 restructuring UL55 albumen

2.3.1 a large amount of cultivations of thalline

Concrete steps are: (1) is inoculated in the expression bacterium E.coli BL21 (DE3) containing pET32a-UL55 plasmid in 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 ℃, under the condition of pH 7.0, cultivate bacterium liquid OD to be cultured to ₆₀₀during=0.6 left and right, adding IPTG is 0.2mmol/L to final concentration, and 4h is cultivated in 37 ℃ of inductions; (4) collect cultured bacterium liquid (about 5L), after the centrifugal 10min of 8000r/min, collect bacterial sediment, resuspended with appropriate Tris-HCl (20mmol/L, pH 8.0) after, adding lysozyme to make its final concentration is 1mg/ml ,-20 ℃ save backup.

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

Take out the bacterial sediment of-20 ℃ of preservations, after melting under 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 by 20ml washing lotion (10mmol/LPBS+2mol/L urea+1%Triton X-100+20mM Tris-HCl+2mM EDTA) and suspend, after 4 ℃ of centrifugal 10min of 10000r/min, precipitation suspends by 20ml washing lotion again, after repeated washing five times, with appropriate urea liquid (10mmol/LPBS+8M urea+50mM Tris-HCl+50mM NaCl+10% glycerine) dissolution precipitation, 4 ℃ save backup.

2.3.3 recombinate renaturation and the detection of UL55 albumen

Inclusion body is washed to the restructuring UL55 albumen that purifying obtains to be joined in 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 control final concentration of protein in the scope of 0.1～1mg/ml.4 ℃ of renaturation 24～48h, the protein liquid of collection dilution refolding, packs 4 ℃ of dialysis of bag filter (dialysis buffer liquid: 50mM Tris-HCl pH 8.0+50mM NaCl+0.5mM EDTA+10% glycerine+1% glycocoll) 24～48h into.After dialysis, sample is collected supernatant after the centrifugal 15min of 8000g, get wherein 20 μ l and carry out SDS-PAGE analysis, and take purifying the anti-DPV of rabbit be primary antibodie, the HRP mark goat anti-rabbit igg of take anti-carries out Western blotting detection as two.All the other protein liquids are measured the ultimate density of albumen by Bradford method, packing postlyophilization is concentrated standby.

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

3 experimental results

The amplification of 3.1DPV UL55 gene, T-cloning and identification result

3.1.1UL55 the pcr amplification result of gene

The DPV CHv strain genomic DNA of take carries out pcr amplification to UL55 gene as template, its product is through 1.0% agarose gel electrophoresis, obtained the specific DNA band of a treaty 799bp, and take normal DEF genomic DNA, carry out pcr amplification as template, without specific band, this consistent with expected results (Fig. 1-A).

3.1.2UL55 gene T clone identification result

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

The Construction and identification of 3.2 prokaryotic expression plasmid pET32a-UL55, abduction delivering and optimum results thereof

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

To reclaim object segment after BamH I and 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 after BamH I and Xho I double digestion is about respectively 5370bp and 799bp (seeing Fig. 1-C), conform to theoretical value, show that prokaryotic expression carrier is successfully built.

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 to expression strain BL21 (DE3) and screening white colony containing on the LB agar plate of Amp.Expressive host bacterium BL21 (DE3) containing recombinant plasmid pET32a-UL55 is carried out to abduction delivering, with IPTG induction, empty carrier pET-32a (+), do not transform bacterial strain abduction delivering with IPTG, and result shows: specific proteins band does not all appear in empty carrier pET-32a (+) conversion bacterial strain inducing and not inducible strain; The restructuring UL55 albumen that recombinant expression plasmid pET32a-UL55 expresses is at 40KD place (Fig. 2-A).

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 processed, electrophoresis result shows: expressing protein is mainly present in precipitation, illustrates that recombinant expression protein exists with insoluble inclusion body form in a large number in thalline.

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 to make its final concentration be respectively 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L induction cultivation 4h, result shows: do not add the control tube of derivant without specific proteins band; With increasing of IPTG concentration, protein induced amount does not have significant difference (Fig. 2-B).But comparatively speaking,, when IPTG concentration is 0.2mmol/L, expressing quantity is slightly high.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 approximately 0.4 o'clock, get 3 sterilizing test tubes, packing 5ml/ pipe, add respectively IPTG to final concentration be 0.2mmol/L, be placed in respectively 25 ℃, 30 ℃, 37 ℃ inductions and cultivate 4h, result: temperature is in the time of 25 ℃, inducible protein amount is less, in the time of 37 ℃ the highest (Fig. 2-C), illustrate that protein induced amount increases gradually along with temperature raises.Therefore, selecting 37 ℃ of temperature is best inducing temperature.

3.2.3.3 the optimization of induction time: be 0.2mmol/L in IPTG concentration, under 37 ℃ of conditions, adopt the different induction times of 4～8h to carry out abduction delivering, result is along with the recombinant protein table amount of the increase induction generation of time reduces.The expression of recombinant proteins amount of induction 5～8h is all significantly lower than 4h induction group; And induction 6～8h, its expression of recombinant proteins amount is lower than 5h and compare to each other without significant change (Fig. 2-D).Therefore, select 4h as best induction time.

3.3 the purification result of restructuring UL55 albumen

By a large amount of expansion, cultivate, collected the bacterial sediment that contains in a large number restructuring UL55 albumen, through lysozyme lysis, ultrasonication, washing and dissolving inclusion body, the processes such as metaprotein dialysis renaturation have obtained the restructuring UL55 albumen of large-scale purification, by SDS-PAGE, analyze and show that the restructuring UL55 albumen of purifying has higher purity (Fig. 3-A), Western blotting analyzes and shows that this restructuring UL55 albumen can strong immune response (Fig. 3-B) occur with anti-DPV positive serum, show that this recombinant protein has higher immunogenicity, can be used as the detectable antigens that detects clinically Antibody To Duck Plague Virus.

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

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

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

Determining of the 1.1 coated concentration of restructuring UL55 albumen and serum dilution

Adopt square formation method to determine best antigen coated concentration and serum dilute concentration, restructuring UL55 albumen after the renaturation that is 4mg/ml to concentration with coating buffer carries out serial dilution (1: 10, 1: 20, 1: 40, 1: 80, 1: 160, 1: 320), coated enzyme reaction plate, every hole is coated with 100 μ l, parallel coated two row of each titre, duck serum (anti-DPV positive serum or nonimmune duck negative serum) is pressed 1: 10, 1: 20, 1: 40, 1: 80, 1: 160, the dilutability of 1: 320 carries out serial dilution, goat-anti duck IgG-HRP does dilution in 1: 2000, with indirect ELISA method, measure, select the OD of positive serum _450nmvalue approaches 1.0, the OD of negative serum _450nmbe worth littlely, the UL55 albumen that 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.

Restructuring UL55 proteantigen is done after serial dilution with coating buffer, then adds 1: 10-1: positive serum or the negative serum of 320 dilutions; The goat-anti duck IgG-HRP ELIAS secondary antibody that separately adds dilution in 1: 2000, adds substrate nitrite ion, by full-automatic microplate reader, measures OD _450nmvalue.The coated concentration of purifying antigen is between 0.4mg/ml～0.0125mg/ml time, and the dilutability of positive serum is 1: 10-1: 320 o'clock, and the OD of different dilution antibody _450nmvalue is in Table 1.When P/N value is 9.667 to the maximum, while being 0.2mg/ml (antigen diluent degree is 1: 20) by the antigen coated concentration of the best of square formation titration gained, the dilutability of positive serum is 1: 160.P/N value corresponding to each antigen-antibody concentration is in Table 2.

The OD of table 1 square formation titration _450nmresult

Determining of the best coated concentration of table 2 DPV-UL55 expressing protein and serum-concentration (P/N value)

Determining of 1.2 ELIAS secondary antibody optimum dilution degrees

The restructuring UL55 proteantigen of purifying is coated in to ELISA reaction plate by the concentration of 0.2mg/ml, every hole 100 μ l, then add positive serum or the negative serum diluting at 1: 160.Afterwards enzyme labelled antibody is done and within 1: 500,1: 1000,1: 2000,1: 4000, after serial dilution, caught ELISA mensuration, detect normal cell culture simultaneously and substitute the OD that duck plague virus makes negative control _450nmvalue, while selecting P/N value maximum, enzyme labelled antibody concentration is as the suitableeest working concentration.Dilution in the present embodiment is all diluted according to volume ratio.

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

The trace routine of the definite and UL55-ELISA method of 1.3UL55-ELISA yin and yang attribute critical value

Get 30 parts of nonimmune duck negative serums, parallel coated 1 time of each sample.The coated concentration of restructuring UL55 albumen, serum to be checked and ELIAS secondary antibody optimum dilution degree are carried out to indirect ELISA mensuration, to determine duck serum its absorption value scope when infecting without DPV.With 1.0%BSA/PBS solution, make blank simultaneously.By the OD of 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 negative.

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

20 parts of DPV negative serum testing results of table 3

The trace routine of 1.4UL55-ELISA method

According to above-mentioned optimum results, the trace routine of indirect ELISA method is as follows: the preparation of (1) solid phase antigen: restructuring UL55 albumen is coated in ELISA Plate to 100 μ l/ holes with 0.2mg/ml concentration, put 4 ℃ of wet box overnight incubation, wash 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 plate machine washing and wash 1 time, 5min/ time, pat dry; (2) one anti-bindings: serum to be checked is added to ELISA Plate after by the volume dilution degree dilution of 1: 160,100 μ l/L, 37 ℃ of wet boxes are hatched 1h, wash plate machine washing and wash 3 times, 5min/ time, pat dry; (3) two anti-bindings: with ELIAS secondary antibody dilution, ELIAS secondary antibody (goat-anti duck IgG-HRP) is added after with the volume dilution degree dilution of 1: 2000,100 μ l/ holes, 37 ℃ of reaction 45min, wash 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, after 25 ℃ of lucifuge colour developing 30min, add 50 μ l/ hole 2mol/L sulfuric acid cessation reactions; (5) detect and judge: by microplate reader, surveying OD _450nmvalue, when microplate reader is surveyed OD _450nmvalue, OD _450nmit is 0.330 positive that value is greater than, be less than or equal to 0.330 negative.With 1.0% BSA/PBS solution, make parallel blank simultaneously.

2 beneficial effect evaluations

2.1 sensitivity experiments

Restructuring UL55 albumen is coated with by the coated concentration of the best, it is that the positive serum of 1: 8 is done 1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800,1: 25600 that 14d after DPV attenuated vaccine immunity, neutralization are tired, 1: 51200 totally 9 volume dilution degree, all the other conditions are carried out UL55-ELISA test by the present embodiment condition, and result is as shown in table 4.When positive serum is diluted to 1: 6400, by visual inspection change color, be difficult to judgement feminine gender, positive findings, but still can detect by microplate reader, press the positive serum testing result of dilution in 1: 12800 lower than critical value 0.330, show that the method can detect the positive serum of 14d after 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 specific test results

Adopt the ELISA condition of establishing, the coated ELISA ELISA Plate of UL55 albumen with purifying, with each two parts of DPV positive serum, DHV positive serum, RA positive serum, E.coli positive serum, Salmonella positive serum, a swollen property haemorrhage positive serum, influenza positive serums, carry out UL55-ELISA specific test, result is as shown in table 5.According to critical value criterion, 2 parts of DPV positive serum OD _450nmbe 0.925,0.896, all much larger than 0.330, be judged to the positive, all the other each virosis or bacterial diseases, duck serum OD _450nmvalue is all less than critical value, all negative, shows that the UL55-ELISA method of the detection DPV antibody of foundation has good specificity.

Table 5 specific test result

P is positive, and N is negative;

2.3 blocking-up property tests

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

Table 6 blocking-up property test findings

3, discuss

In ELISA method testing process, antigen coated concentration is larger on test findings impact, if antigen coated concentration is high, because acting force between antigen protein molecule is compared with causing greatly the multiple stratification (stacking efect) of protein molecular, be easily washed non-specific increase, if concentration is too low, ELISA Plate surface may leave not adsorption antigen but complete totally enclosed active surface, non-specificly also can increase, therefore must screening coating protein antigen concentration.In addition, the purity of antibody is directly connected to specificity and the susceptibility of ELISA test, the antibody that purity is not high usually has the host cell macromolecular compound of being combined with specific antigen of excess, carrier surface position that can be limited with antigenic competition and reduce effective adsorption rate, therefore highly purified antibody protein can improve atopic, but indirect ELISA method detects the antibody in serum conventionally, serum composition is complicated, in production, conventionally one by one tested serum is not carried out to purifying, only have by groping suitable serum diluting multiple, could reduce non-specific binding.This experiment square formation method is determined the optimum diluting multiple of serum (primary antibodie), from the results of view, though serum diluting multiple from 1: 10 to 1: 320, serum-concentration reduces, OD _450nmvalue diminishes, according to OD _450nmvalue approaches the optimum dilution degree that 1.0, P/N maximum is judged antigen-antibody.Therefore definite antigen coated concentration of the best is 0.2mg/ml, and the extension rate of serum is 1: 160.

The height of ELIAS secondary antibody concentration also has larger impact to test findings, and concentration is too high, and the chance of non-specific binding increases, and may occur false positive, and concentration is too low, there is no effective combination, may occur the phenomenons such as false negative.This experiment is carried out the optimum diluting multiple that indirect ELISA is determined ELIAS secondary antibody under the ELISA condition of having optimized.According to OD _450nmvalue approaches 1.0, P/N maximum and judges optimum dilution degree, and the optimum diluting multiple of determining ELIAS secondary antibody is 1: 2000.Sealing is the irrelevant protein solution coated process again of use high concentration after coated, concentration used when antigen coated is lower, after absorbing, surface of solid phase carriers still has the space not being occupied, sealing is exactly to allow these spaces of a large amount of incoherent protein fillings, thereby repels adsorbing again of in ELISA step thereafter interfering material.The most frequently used sealer is 0.5%-1.0% bovine serum albumin(BSA), and also useful skimmed milk power or 1.0% gelatin are as sealer.Skimmed milk power is also a kind of good sealer, and its maximum feature is inexpensive, but due to the complicated of milk powder, and the carrier after sealing is difficult for long-term preservation, therefore less application in the preparation of kit.In the present embodiment, select 1.0% BSA to make confining liquid, its composition is single, and sealing effect is better.

In ELISA criterion, conventional two kinds of methods, a kind of is with the original OD that measures sample _450nmvalue and standard variance are determined critical value.Another kind is to calculate testing sample with respect to the ratio of negative control, and the frequency of tiring by mensuration great amount of samples distributes and determines critical value and suspicious interval.This research, when definite yin and yang attribute criterion, has adopted the first criterion.According to the OD of a large amount of negative samples _450nmvalue, adopts statistical method to determine negative and positive critical value.The principle of foundation is the OD of sample _450nmvalue > negative sample OD _450nmvalue mean value (X)+3SD time, can in 99.9% level, be judged to the positive, according to principle of statistics, great many of experiments has confirmed that this criterion is reliable.In addition, when the reports such as Alonso utilize the albumen of Bacillus coli expression to detect field pig serum as envelope antigen, the problem that may exist host protein to disturb, the Escherichia coli antibody existing in pig serum likely with expressing protein in remaining host protein generation nonspecific reaction.Therefore, in setting up the process of UL55-ELISA method, selected the E.coli positive serum in duck source to do specificity experiment, result shows this UL55-ELISA method and E.coli serum no cross reaction, and specificity is good.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, do specific test again simultaneously, result has all confirmed that the UL55-ELISA setting up has good specificity and susceptibility, shown good application prospect, also for being further assembled into kit, laid a good foundation.

Embodiment 3

Another example, the trace routine of indirect ELISA method is as follows: the preparation of (1) solid phase antigen: restructuring UL55 albumen is coated in ELISA Plate to 100 μ l/ holes with 0.2mg/ml concentration, put 4 ℃ of wet box overnight incubation, wash 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 plate machine washing and wash 1 time, 5min/ time, pat dry; (2) one anti-bindings: serum to be checked is added to ELISA Plate after by the volume dilution degree dilution of 1: 20,100 μ l/L, 37 ℃ of wet boxes are hatched 1h, wash plate machine washing and wash 3 times, 5min/ time, clap in; (3) two anti-bindings: with ELIAS secondary antibody dilution, ELIAS secondary antibody (goat-anti duck IgG-HRP) is added after with the volume dilution degree dilution of 1: 1000,100 μ l/ holes, 37 ℃ of reaction 45min, wash 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, after 25 ℃ of lucifuge colour developing 30min, add 50 μ l/ hole 2mol/L sulfuric acid cessation reactions; (5) detect and judge: by microplate reader, surveying OD _450nmvalue, when microplate reader is surveyed OD _450nmvalue, OD _450nmit is 0.330 positive that value is greater than, be less than or equal to 0.330 negative.With 1.0% BSA/PBS solution, make parallel blank simultaneously.

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

Claims

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

The preparation of a solid phase antigen: the restructuring UL55 protein liquid solid phase carrier that is 0.2mg/ml by coated concentration connects, with confining liquid sealing, unconjugated antigen and impurity are removed in washing, obtain solid phase antigen; Described solid phase carrier is ELISA Plate; Described confining liquid is that volumetric concentration is 1.0% bovine serum albumin(BSA);

C bis-anti-bindings: ELIAS secondary antibody is done to the dilution of 2000 times, obtained ELIAS secondary antibody dilution, described ELIAS secondary antibody dilution is combined with described solid phase antigen antibody complex, obtain the anti-compound of Ag-Ab-bis-; Described ELIAS secondary antibody is goat-anti duck IgG-HRP;

D colour developing: add after the colour developing of chromogen substrate lucifuge at the anti-compound of step c gained Ag-Ab-bis-, add the stop buffer cessation reaction sample liquid that must develop the color; Described chromogen substrate is tetramethyl benzidine;

E detects and judges: by developing the color described in steps d, sample liquid is surveyed OD by microplate reader _450nmvalue, described OD _450nm>0.330 is positive for value, described OD _450nm≤ 0.330 negative; The time of lucifuge colour developing is 30 minutes;

The addition of ELIAS secondary antibody dilution described in dilute serum described in UL55 protein liquid, step b and step c of recombinating described in step a is equal-volume and is more than or equal to 100 μ l.

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