CN102012428B

CN102012428B - Colloidal gold test paper based on recombinant UL51 protein resisting antibody, preparation method and application thereof

Info

Publication number: CN102012428B
Application number: CN 201010299524
Authority: CN
Inventors: 程安春; 汪铭书; 沈婵娟; 陈孝跃
Original assignee: ENGINEERING AND TECHONLOGY CENTER FOR LABORATORY ANIMALS OF SICHUAN AGRICULTURAL UNIVERSITY
Current assignee: Sichuan Agricultural University
Priority date: 2010-09-30
Filing date: 2010-09-30
Publication date: 2013-09-25
Anticipated expiration: 2030-09-30
Also published as: CN102012428A

Abstract

The invention relates to colloidal gold test paper based on a recombinant UL51 protein resisting antibody, which comprises an absorption pad, a bottom plate, a nitro fiber membrane, a colloidal gold pad and a sample pad, wherein the testing line on the nitro fiber membrane is formed by coating an A animal recombinant UL51 protein resisting antibody IgG with concentration of not less than 1mg/mL, the colloidal gold pad is formed by coating a colloidal gold label B animal recombinant UL51 protein resisting antibody IgG with concentration of not less than 2mg/mL, and the contrast line on the nitro fiber membrane is formed by coating a C animal resisting B animal IgG with concentration of not less than 1mg/mL. A preparation method of the colloidal gold test paper comprises the steps of: spraying the testing line and the contrast line on the nitro fiber membrane, preparing the colloidal gold pad, and assembling a colloidal gold immunochromatographic test strip. The test paper is used for detecting duck plague virus antigens, and has high detection sensitivity and high specificity. The preparation method is simple and practical in operation; and the recombinant UL51 protein resisting antibody is prepared into the colloidal gold test paper for detecting the duck plague virus antigens.

Description

Based on colloid gold test paper of anti-reorganization UL51 protein antibodies and its preparation method and application

Technical field

The present invention relates in the animal medicine field duck plague virus detection of antigens and detect, particularly based on colloid gold test paper of anti-reorganization UL51 protein antibodies and its preparation method and application.

Background technology

Duck plague (Duck plague, DP), claim again duck viral enteritis (Duck viral enteritis, DVE), be a kind of acute contagious disease of Anseriformes animals such as duck, goose, swan, its pathogenic feature is injury of blood vessel, it is hemorrhage to organize, alimentary canal and lymphoid organ damage.This disease can cause the egg production of commodity aquatic bird to descend and be dead, and wild aquatic bird is also had different fatal rates.The cause of disease of DP be DPV (Duck plague virus, DPV), DPV is a kind of virus of pantropic systemic infection, at present Most scholars is temporarily classified it as Alphaherpesvirinae, but does not adhere to separately as yet.At present, the DPV detection method of having reported has virus to separate evaluation, PCR (PCR), fluorescence real-time quantitative PCR, indirect immunoperoxidase group method, indirect immunofluorescence, electron microscopic observation, in situ hybridization, indirect in situ PCR method, serum neutralization test (SNT), agar gel diffusion test, enzyme linked immunosorbent assay (ELISA), reversed passive hemagglutination test, micro solid phase radioimmunoassay method, biotin labeling oligonucleotide probe in situ detection, digoxigenin labeled nucleic acid probe method and photobiotin labelling method etc., and these methods respectively have characteristics.But traditional viral isolation and identification method approximately needs 4～5d, and method is loaded down with trivial details, wastes time and energy.Some immunological methods and PCR method also often need special instrument, equipment and those skilled in the art.In addition, the antibody that many serology detection methods all are based on the DPV totivirus generation of purifying detects DPV, owing to can be doped with many host cell compositions in the complicacy of viral purification and the virus behind the purifying, and cause many false positive results to occur.

Collaurum (ICS) method is a kind of immobilon-p immune analysis method that colloid gold immune technology and chromatography technology are combined that grows up the eighties in 20th century, and it develops on immune colloid gold percolation basis.Obtained increasingly extensive application in biomedical each field in recent years, as the detection of parasitic disease, virosis, bacterial disease and medicament residue etc.Simple to operate, the high specificity of this method, and can in 15min, judge having or not of determinand very intuitively, improved the speed that detects greatly.The ICS method has now become one of current quick, the most responsive immunology detection technology, be particularly suitable for the vast animal doctor personnel of basic unit and mass detection and large tracts of land generaI investigation etc., so this technology has huge development potentiality and application prospect.Along with molecular biological develop rapidly, research to protein function strengthens day by day, particularly increasing to the research that immunogenic protein is arranged, detect viral sero-fast ELISA method as coating antigen and colloidal gold immunity chromatography is reported in a large number with the recombinant protein of clonal expression, the report of the antibody test viral antigen of the recombinant protein preparation expressed is also arranged simultaneously.But, do not appear in the newspapers as yet based on the ICS method of the antibody test duck plague virus antigen of reorganization UL51 albumen and whether with DPV the strong immune response confirmation of still needing to take place to, the antibody of reorganization UL51 albumen.

Summary of the invention

In view of this, one of purpose of the present invention is to provide a kind of colloid gold test paper, this test paper is based on anti-reorganization UL51 protein antibodies mark and strict control gold mark pad and goes up on the basis of the label concentration of antibody on immune colloid gold and the nitrocellulose film and be prepared from its specificity and sensitivity height.

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

Colloid gold test paper based on anti-reorganization UL51 protein antibodies, formed by base plate, nitrocellulose film, sample pad, gold mark pad and absorption pad, p-wire and control line are arranged on the nitrocellulose film, p-wire on the described nitrocellulose film is formed more than or equal to the anti-reorganization of the A of 1mg/mL UL51 protein antibodies IgG bag by concentration, and control line is formed more than or equal to the anti-BIgG bag of 1mg/mL C by concentration; Described gold mark pad is formed more than or equal to the anti-reorganization of the B of 2mg/mL UL51 protein antibodies IgG bag by colloid gold label concentration; The anti-reorganization of described A UL51 protein antibodies IgG is the anti-reorganization of any animal UL51 protein antibodies IgG except duck in the normal experiment animal in the immunology research, the anti-reorganization of described B UL51 protein antibodies IgG is the anti-reorganization of any animal UL51 protein antibodies IgG except A in the normal experiment animal in the immunology research, and the anti-B IgG of described C is the anti-B IgG of any animal except A and B in the normal experiment animal in the immunology research.

Further, the A among the anti-reorganization of the described A UL51 protein antibodies IgG is mouse-anti reorganization UL51 protein antibodies IgG, and the anti-reorganization of described B UL51 protein antibodies IgG is the anti-reorganization of rabbit UL51 protein antibodies IgG, and the anti-B IgG of described C is goat anti-rabbit igg;

Further, the p-wire on the described nitrocellulose film is formed by the anti-reorganization of the A UL51 protein antibodies IgG bag that concentration equals 1mg/mL, and control line equals the anti-B IgG bag of 1mg/mL C by concentration and formed; Described gold mark pad bag is formed by the anti-reorganization of the B UL51 protein antibodies IgG bag that colloid gold label concentration equals 2mg/mL;

Two of purpose of the present invention is to provide the preparation method of described colloid gold test paper, and this method is simple and practical.

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

The method for making of above-mentioned colloid gold test paper based on anti-reorganization UL51 protein antibodies, concrete steps are:

The spray of p-wire and control line on a nitrocellulose film: concentration is formed p-wire more than or equal to the anti-reorganization of the A of 1mg/mL UL51 protein antibodies IgG specking on the nitrocellulose film, concentration is formed control line more than or equal to the anti-B IgG of 1mg/mLC specking on the nitrocellulose film, dry back low temperature seal is preserved standby;

The preparation of the golden mark pad of b: the plain film of glass fibre is dipped in colloid gold label concentration more than or equal among the anti-reorganization of the B of the 2mg/mL UL51 protein antibodies IgG, again this film is dipped in the plain film treating fluid of glass fibre and seals nonspecific binding site, get golden mark pad after the drying;

The assembling of c colloidal gold immuno-chromatography test paper strip: respectively nitrocellulose film, sample pad, gold mark pad and absorption pad are bonded on the described base plate successively, the above control line of nitrocellulose film is near the absorption pad end, described p-wire is near the sample pad end, cut into the test strips of certain width again, pack, dry low temperature is preserved.

Further, the distance of described p-wire and described control line is 0.7 centimetre, and described p-wire and described control line are respectively 0.9 centimetre apart from the back gauge of nitrocellulose film.

Three of purpose of the present invention is to provide the utilization of described colloid gold test paper, and described utilization can guarantee fast detecting duck plague virus antigen under sensitivity and specificity condition with higher.

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

The application of colloid gold test paper in preparation duck plague virus antigen detection test paper.

Beneficial effect of the present invention is: this colloid gold test paper susceptibility height, and can detect minimum content is the DPV virus protein of 0.125 μ g; Use this test paper to the DEF nutrient solution, the duck embryo allantoic liquid that contains DHV that contain DPV, contain the bacterial culture fluid of RA and contain the E.coli bacterial culture fluid, the result shows visible two red stripes clearly of the DEF nutrient solution only contain DPV, a red stripes clearly all only appears in other sample liquid at C line place, show that the method has good specificity; In addition, use this detection paper duck plague virus to have in good batch and batch between repeatability; The test paper of this method preparation can be preserved half a year at 4 ℃ or 25 ℃ at least, and this utilization will resist reorganization UL51 protein antibodies IgG to be prepared into colloid gold test paper for detection of duck plague virus first.

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, wherein:

Fig. 1-A is that the pcr amplification of DPV UL51 gene: M refers to DL2000 relative molecular mass standard, the pcr amplification product that 1 finger is 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 760bp) of template; Fig. 1-B is that the double digestion of recombinant plasmid pMD18-UL51 is identified: M refers to relative molecular mass standard I II, and 1 refers to two fragments (the molecular weight size of small fragment shown in the arrow is about 760bp) that recombinant plasmid pMD18-UL51 obtains with EcoR I and Xho I double digestion; Fig. 1-C is that the double digestion of recombinant expression carrier pET28a-UL51 is identified: M refers to relative molecular mass standard I II, 1 refers to recombinant expression carrier pET28a-UL51, and 2 refer to two fragments (the molecular weight size of small fragment shown in the arrow is about 760bp) that recombinant expression carrier pET28a-UL51 obtains with EcoR I and Xho I double digestion.

Fig. 2-A is that the SDS-PAGE of recombinant expression protein identifies: M is protein relative molecular mass standard, 1 negative contrast (not adding IPTG induces), and 2 induce (the protein molecular weight size shown in the arrow is about 34KD) for IPTG; Fig. 2-B is the different final concentration abduction delivering of derivant IPTG result: the IPTG concentration of 1-7 is respectively 0,0.2,0.4,0.6,0.8,1.0 and 1.2mmol/L; Fig. 2-C is that different temperatures induces the temperature of pET28a-UL51 expression of results: 1-3 to be respectively 20,30 and 37 ℃; Fig. 2-D is that different time induces the induction time of pET28a-UL51 expression of results: 1-7 to be respectively 1,2,3,4,5,6,7 and 8h.

Fig. 3-A analyzes for the SDS-PAGE of reorganization UL51 protein purification product: M is protein molecular weight, the 1 1ml bacterium liquid of inducing for IPTG, and 2 UL51 albumen inclusion bodys for slightly carrying, 3 is 50mM imidazoles eluting peak (the reorganization UL51 albumen that does not contain purifying); 4 are 300mM imidazoles eluting peak (the reorganization UL51 albumen that contains purifying); Fig. 3-B is that the SDS-PAGE of the anti-reorganization UL51 protein antibodies IgG of purifying analyzes: the accurate protein molecular weight of M index, and 1 referred to the anti-UL51 protein antibodies of the rabbit IgG of post purifying, 2 was post purifying mouse-anti UL51 protein antibodies IgG.

Fig. 4 detects the titration of the anti-reorganization of rabbit UL51 albumen hyper-immune serum for the Ago-Gel diffusion test.

Fig. 5 is the assembling synoptic diagram of colloidal gold immuno-chromatography test paper strip;

Fig. 6 is the sensitivity Detection result based on the ICS method of anti-reorganization UL51 protein antibodies.

Embodiment

In order to make the purpose, 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.In the present embodiment, test paper is based on anti-reorganization UL51 protein antibodies mark and strict control gold mark pad and goes up on the basis of the label concentration of antibody on immune colloid gold and the nitrocellulose film and be prepared from, the acquisition of described reorganization UL51 albumen is by making up prokaryotic expression plasmid pET28a-UL51, transforming and express bacterium and fermented and cultured, collected a large amount of bacterial sediments that contains reorganization UL51 albumen, obtained by ultrasonic disruption thalline, reorganization UL51 albumen inclusion body Xian Di, purifying and gradient dialysis again; With described reorganization UL51 albumen rabbit and mouse are carried out routine immunization program and purifying, obtain rabbit anti-reorganization UL51 protein antibodies IgG and mouse-anti reorganization UL51 protein antibodies IgG; Further determine optimum concentration range and the enzyme labelled antibody optimum concentration of rabbit anti-reorganization UL51 protein antibodies IgG and mouse-anti reorganization UL51 protein antibodies IgG, finally obtained the high detection test paper of specificity and sensitivity.

Embodiment is based on colloid gold test paper of anti-reorganization UL51 protein antibodies and its preparation method and application

The purifying of the clone of one duck plague virus UL51 gene, prokaryotic expression and UL51 albumen

1 material is prepared

1.1 bacterial strain, plasmid and strain

Plasmid pMD18-T is available from the precious bioengineering in Dalian company limited; Prokaryotic expression plasmid pET28a (+), 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 experimental animal

10 age in days duck embryos, its kind duck DPV and antibody are all negative; 4 of healthy male rabbits, body weight 2-3kg/, available from the warren, Yaan; 20 of healthy Vista rats are available from Sichuan University.

2 experimental techniques

2.1DPV the clone of UL51 gene

2.1.1 design of primers

Utilize Primer Premier5.0 software, with reference to UL51 gene order (GenBank accession number: DQ072725), synthetic by precious biotinylated biomolecule technology company limited.

Primer DPV-UL51F:5 '-CCG GAATTCATGTTAGCTTTTATCTCCAG-3 ' (the line part is the EcoRI site); Primer DPV-UL51R:5 '-TCC CTCGAGTTAGACGGCTACCAACG-3 ' (the line part is the XhoI 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

The method for making of the normal DEF of a (DEF): 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 with PBS idiosome is cleaned, 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 (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, add in the filtrate 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.

B 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.

C DNA extracting 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 with the saturated phenol washing of 500 μ L, pour in the centrifuge tube; (4) use 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/LNaAC, 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 UL51 gene

The PCR reaction system is:

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

Response parameter: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate 40 times, and last 72 ℃ are extended 10min, standby in 4 ℃ of preservations.Get 4 μ L PCR products electrophoresis on 1% Ago-Gel, establish DL2000 and blank, observe the length of amplified fragments.

2.1.4UL51 the recovery of gene PCR product

Reclaim the kit instructions by Beijing match Parkson DNA of gene technology company limited and carry out, it is standby that the DNA after the recovery is stored in-20 ℃ of preservations.

2.1.5 the UL51 gene of purifying and being connected of pMD18-T

The coupled reaction system is as follows:

Mentioned reagent is added in the EP pipe of 0.2mL, careful mixing, instantaneous centrifugal after, spend the night in 16 ℃ of connections.

2.1.6DH5a the preparation of competent cell

Adopt Calcium Chloride Method to prepare fresh DH5a strain competent escherichia coli cell, be summarized as follows: the DH5a monoclonal colony inoculation of fresh cultured spends the night in 37 ℃ of shaken cultivation in 5mL LB nutrient solution on (1) aseptic picking flat board; (2) get the above-mentioned nutrient solution of 1mL and be inoculated in the 100mL LB nutrient solution, 37 ℃ of 200r/min shaken cultivation 2.5～3h make OD ₆₀₀About=0.5; (3) bacterial cultures is poured in the ice-cold centrifuge tube in sterilization back into ice bath 10min; (4) in 4 ℃ of centrifugal 8min of 4000r/min, abandon supernatant; (5) add the CaCl that 10mL ices the 0.1mol/L of precooling ₂, the gentle bacterial precipitation, ice bath 30min of having hanged; (6) in 4 ℃ of centrifugal 8min of 4000r/min, abandon supernatant, add the CaCl of the 0.1mol/L of 4mL ice precooling ₂Resuspended precipitation again adds final concentration and is 15% sterile glycerol, is packed as 200 μ L/ pipe behind the mixing, is directly used in to transform or to place-70 ℃ of refrigerators to preserve standby.

2.1.7 transformed competence colibacillus cell

The whole taking-up of 10 μ L linked systems is added in the 200 μ L DH5a competent cells ice bath 30min; Place 42 ℃ of temperature to bathe 90s again, afterwards ice bath 2min again; Add 0.8mL LB nutrient culture media then immediately, 37 ℃ of water-bath shaken cultivation 45min get 200 μ L and are applied on the nutrient culture media that contains (X-gal/IPTG/Kan), put overnight incubation in 37 ℃ of incubators.The single white colony of picking next day is inoculated among the 5mL LB (containing 50 μ g/mL Kan), carries out plasmid extraction behind 37 ℃ of water-bath shaken cultivation 18h.

2.1.8 the extracting of plasmid

Undertaken by match Parkson, Beijing gene technology company limited plasmid extraction kit instructions, it is standby that the recovery product is stored in-20 ℃ of preservations.

2.1.9 PCR and the enzyme of recombinant plasmid are cut evaluation

With the recombinant plasmid called after pMD18-UL51 of previous step extracting, respectively with the digestion of EcoR I/XhoI double digestion and the digestion of Xho I single endonuclease digestion, 1.0% gel electrophoresis observations.Do the pcr amplification genes of interest simultaneously.

2.1.10UL51 gene sequencing

Send the biological company limited of Shanghai English fine horse and check order identifying correct plasmid.

2.2 the structure of prokaryotic expression plasmid pET28a-UL51, abduction delivering and expression condition optimization

2.2.1 the structure of prokaryotic expression plasmid pET28a-UL51 and evaluation

The enzyme of a purpose fragment is cut and is connected: restriction enzyme EcoR I and Xho I be double digestion pMD18-UL51 plasmid and prokaryotic expression carrier pET28a (+) 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.

The conversion of b recombinant plasmid: adopt Calcium Chloride Method to prepare the DH5a competent cell.Afterwards, get connection liquid 15 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, then rapid ice bath 2min; Add the LB fluid nutrient medium 800 μ L that do not conform to Kan, 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 Kan, 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.

The enzyme of c 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 Kan 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, EcoR 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 described primer to carry out the PCR reaction, its method and amplification condition are the same, get PCR product electrophoresis detection on 1% Ago-Gel.Cut the evaluation with PCR through enzyme, obtain reorganization prokaryotic expression plasmid pET28a-UL51.

2.2.2 the abduction delivering of recombinant expression plasmid pET28a-UL51

The extraction of a recombinant plasmid pET28a-UL51: picking has identified that the DH5a bacterial classification streak inoculation that contains positive recombinant plasmid pET28a-UL51 is on the LB agar plate that contains Kan 50g/mL, 37 ℃ of overnight incubation, get single colony inoculation next day on the 5mLLB fluid nutrient medium, thermal agitation is cultivated 10～16h, centrifugal collection bacterium liquid is pressed UltraPure ^TMPlasmid DNA is extracted the extraction and purification that recombinant plasmid is carried out in the kit explanation in a small amount.

B recombinant plasmid pET28a-UL51 transforms and expresses bacterium: adopt Calcium Chloride Method to prepare E.coli BL (DE3) competent cell, and the recombinant plasmid pET28a-UL51 of said extracted is transformed among the expressive host bacterium E.coli BL (DE3).

The abduction delivering of c recombinant plasmid pET28a-UL51: from above-mentioned LB solid medium (containing Kan 50 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 5mLLB fluid nutrient medium (containing Kan 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 3h, 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.

The soluble analysis of d recombinant plasmid pET28a-UL51 expression product: with the 100mL bacterium liquid of abduction delivering and the 100mL bacterium liquid of abduction delivering not, press step process respectively: 4 ℃, the centrifugal 5min of 10000r/min, bacterial sediment suspends with 20mL 20mmolTris-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 ℃, the centrifugal 10min of 10000r/min, 1. get supernatant standby; Precipitation suspends with 10mL washing lotion (10mmol/L PBS+2mol/L urea+0.2%Triton X-100), 4 ℃, behind the centrifugal 10min of 10000r/min, precipitation suspends with the 10mL washing lotion again, behind the repeated washing three times, with an amount of urea liquid (10mmol/L PBS+8mol/L urea) dissolution precipitation 2., low temperature is preserved standby.Get respectively an amount of supernatant 1. with the precipitation of urea liquid dissolving 2., 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.And will dye lustful gel and induce in the bacterium liquid recombinant protein relative percentage composition of (precipitating 2. the inclusion body form) in endochylema (supernatant 1., solubility) and precipitation through full automatic gel imaging analysis system scan and Quantity One software analysis.

2.2.3 the optimization of recombinant plasmid pET28a-UL51 expression condition

The concentration optimization of a derivant IPTG: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET28a-UL51, in the inoculation 5mL LB fluid nutrient medium (containing Kan 50 μ g/mL), 37 ℃ of jolting overnight incubation.Changeed being inoculated in the 5mLLB fluid nutrient medium (containing Kan 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 7 test tubes, add respectively IPTG to final concentration be after 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L induce for 37 ℃ and cultivate 4h, by described method sample is handled, the 12%SDS-PAGE electrophoresis, observations.

The b temperature conditions is optimized: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET28a-UL51, and in the inoculation 5mL LB fluid nutrient medium (containing Kan 50 μ g/mL), 37 ℃ of jolting overnight incubation.Changeed being inoculated in the 5mL LB fluid nutrient medium (containing Kan 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 0.8mmol/L, place 20 ℃, 30 ℃, 37 ℃ to induce and cultivate 4h respectively, sample is handled 12%SDS-PAGE electrophoresis, observations by above-mentioned conventional method.

The c induction time is optimized: get the expressive host bacterium BL21 (DE3) that contains recombinant plasmid pET28a-UL51, and on the inoculation 5mL LB fluid nutrient medium (containing Kan 50 μ g/mL), 37 ℃ of jolting overnight incubation.Changeed being inoculated on the 5mL LB fluid nutrient medium (containing Kan 50 μ g/mL) next day by 1: 50, continue to be cultured to OD ₆₀₀Be worth about 0.4 o'clock, add IPTG to final concentration be 0.8mmol/L, induce cultivation for 37 ℃, respectively at inducing back 0,1,2,3,4,5,6,7,8h, draw the 1mL nutrient solution, as stated above sample is handled 12%SDS-PAGE electrophoresis, observations.

2.3 a large amount of preparations, purifying and the renaturation of reorganization UL51 albumen

2.3.1 a large amount of preparations (inclusion body processing) of reorganization UL51 albumen

Abduction delivering 1000mL bacterium liquid in 4 ℃, the centrifugal 10min of 10000r/min, is suspended bacterial sediment with 40mL20mmolTris-HCl (pH8.0); Put-20 ℃ of backs of spending the night and add lysozyme by 1mg/mL, 4 ℃ are stirred 30min, and ultrasound wave (ice bath) is broken thalline (200w, 30sec/ time, 5～10 times) intermittently, 4 ℃, the centrifugal 10min of 10000r/min.To precipitate with 20mL washing lotion (10mmol/L PBS+2mol/L urea+0.2%Triton X-100) and suspend, behind 4 ℃, the centrifugal 10min of 10000r/min, with an amount of urea liquid (10mmol/L PBS+8mol/L urea) dissolution precipitation, 4 ℃ of preservations are standby.

2.3.2 the purifying of reorganization UL51 albumen

Nickel Ago-Gel (Ni ²⁺-NAT) affinitive layer purification reorganization UL51 albumen: the affinity interaction special to nickel ion according to the 6-His label of fusion, the fusion that has label can be incorporated on the nickel Ago-Gel, change the condition of eluent with its wash-out, and reach the purpose of purifying.The concrete operations step is: (1) dress post: nickel Ago-Gel dress post, and bed volume is about 40mL; (2) balance: with about 5 the bed volume balance chromatographic columns of level pad I, flow velocity is 1mL/min; (3) go up sample: with the about 5mL of solubility inclusion body sample of 0.45 μ m membrane filtration, add in the chromatographic column, flow velocity is 0.5mL/min; (4) washing: wash 2-5 bed volume again with level pad II, flow velocity is 1mL/min; (5) wash-out: respectively with contain 50,300, the elution buffer III of 500mmol imidazoles carries out gradient elution, flow velocity is 1mL/min, collects the eluting peak of each gradient, detects molecular weight size and the purity of fusion with SDS-PAGE; (4) clean: with 5 bed volumes of ultrapure washing, wash 3 bed volumes with 25% ethanol again, flow velocity is 1mL/min, reclaims nickel Ago-Gel post, preserves in 4 ℃.

2.3.3 the renaturation of reorganization UL51 albumen

To cross the reorganization UL51 albumen of post purifying, gradient dialysis in 4 ℃ of urea liquids at variable concentrations (6,4,3,2,1,0mol/L) makes metaprotein renaturation gradually, and with the ultimate density of Bradford method mensuration albumen, standby after the packing.

3 experimental results

3.1DPV the amplification of UL51 gene, T-clone and qualification result

3.1.1UL51 the pcr amplification result of gene

Be that template is carried out pcr amplification to the UL51 gene with DPV CHv strain genomic DNA, its product is through 1.0% agarose gel electrophoresis, obtained the specific DNA band of a treaty 760bp, 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).

3.1.2UL51 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-UL51.To pMD18-UL51 carry out PCR, enzyme is cut (Fig. 1-B: two fragments that recombinant plasmid pMD18-UL51 obtains with EcoR I and Xho I double digestion, the molecular weight size of small fragment shown in the arrow is about 760bp) and the order-checking evaluation, the result shows that the UL51 gene order that the T clone obtains is in full accord with known DPV UL51 gene order.。

3.2 the structure of prokaryotic expression plasmid pET28a-UL51 and evaluation, abduction delivering and optimization result thereof

3.2.1 structure and the enzyme of recombinant expression plasmid pET28a-UL51 are cut evaluation

To reclaim the purpose segment behind EcoR I and the Xho I double digestion T cloned plasmids, be connected with pET-28a (+) expression vector of cutting through same enzyme, transform DH5 α, obtain recombinant expression plasmid pET28a-UL51, this theory size is about 6130bp, the size of two segments that obtain behind EcoR I and Xho I double digestion is about 5370bp and 760bp respectively and (sees and 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 pET28a-UL51

The abduction delivering of a recombinant plasmid pET28a-UL51 transforms recombinant plasmid pET28a-UL51 and expresses: bacterial strain BL21 (DE3) has screened white colony at the LB agar plate that contains Kan.The expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET28a-UL51 with IPTG carry out abduction delivering, not with IPTG induce, empty carrier pET-28a (+) transforms the bacterial strain abduction delivering, the result shows: empty carrier pET-28a (+) transform bacterial strain inducing and not inducible strain the specific proteins band does not all appear; The reorganization UL51 albumen that recombinant expression plasmid pET28a-UL51 expresses is (Fig. 2-A) at the 34KD place.

The soluble analysis of b recombinant plasmid pET28a-UL51 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.Simultaneously Quantity One software analysis shows: induce in the bacterium liquid recombinant protein at endochylema supernatant (solubility) and in precipitating the relative percentage composition of (inclusion body form) be respectively 6.72% and 93.28%.

3.2.3 the optimization of recombinant plasmid pET28a-UL51 expression condition

The optimization of a, IPTG concentration: under 37 ℃ of conditions, add that IPTG makes its final concentration be respectively 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L induces and cultivates 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 increases gradually, reaches maximum when being increased to the 0.8mmol/L expressing quantity; When increasing IPTG concentration to 1.0mmol/ and 1.2mmol/L thereafter again, there is not significant difference (Fig. 2-B) when its expressing quantity and 0.8mmol/L.Therefore, can select the IPTG concentration of 0.8mmol/L as abduction delivering concentration.

The optimization of b, 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, add respectively IPTG to final concentration be 0.8mmol/L, place 20 ℃, 30 ℃, 37 ℃ to induce and cultivate 4h respectively, the result: temperature is in the time of 20 ℃, 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 ℃.

The optimization of c, induction time: be 0.8mmol/L in IPTG concentration, under 37 ℃ of conditions, adopt the different induction times of 1～8h to carry out abduction delivering, almost do not have special protein band during 1h as a result and produce; Induce the expression of recombinant proteins amount of 1～3h all to be lower than 4h and induce group; Induce 5～8h, its expression of recombinant proteins amount is compared no significant change (Fig. 2-D) with 4h.Therefore, select 4h as best induction time.

3.3 the purification result of reorganization UL51 albumen

After a series of pre-treatments such as the cracking of expression product process lysozyme, ultrasonic disruption, urea washing, nickel Ago-Gel affinitive layer purification reorganization UL51 albumen.The UV curve map that the UL51 protein sample is crossed behind the post purifying demonstrates 3 peaks, and peak 1 is for penetrating the peak, and peak 2 is 50mmol imidazoles eluting peak, and peak 3 is 300mmol imidazoles eluting peak.Collect variable concentrations imidazoles eluting peak simultaneously, carry out the SDS-PAGE electrophoresis, check purity and concentration, the result shows: have only and contain a large amount of highly purified reorganization UL51 albumen (Fig. 3-A) in the 300mmol imidazoles eluting peak.After the reorganization UL51 albumen dialysis renaturation with purifying, use the Bradford method to measure the final concentration of albumen, and its dilution is 1mg/mL, standby after the packing.

Two based on colloid gold test paper of anti-reorganization UL51 protein antibodies and its preparation method and application

Preparation and the purifying of 1 anti-reorganization UL51 protein polyclone antibody

1.1 Freund

The a incomplete Freund: 3 parts of saxols, 1 part in sheep oil mixes, and 115 ℃, gets incomplete Freund behind the 15min autoclaving.Heating and melting before using is cooled to about 50 ℃, adds antigen and carries out emulsification treatment; The b Freund's complete adjuvant: add Bacille Calmette-Guerin on the basis of the above and get final product to final concentration 1mg/mL, with antigenic solution and its mixed in equal amounts, fully emulsified during use.

1.2 preparation and the purifying of the anti-reorganization of rabbit UL51 protein antibodies IgG

1.2.1 the preparation of the anti-reorganization of rabbit UL51 protein antibodies (repeat three these tests, each 1 week at interval is to prepare the antibody of three different batches)

The immunogenic preparation of a: with the reorganization UL51 albumen after purifying and the renaturation, head exempts to mix with the equivalent complete Freund's adjuvant also fully emulsified, and second and third exempts to mix with incomplete Freund's adjuvant also fully emulsified, and the 4th exempts from not add adjuvant.

The b immune programme for children: select 4 of healthy male rabbits, head exempts from the previous day and takes a blood sample respectively, and separation of serum is as negative control.Preparation rabbit anti-UL51 hyper-immune serum immune programme for children such as following table:

Four exempt from back 14d arteria carotis bloodletting, collect blood.Place 1h, 4 ℃ of refrigerator overnight for 37 ℃.Separated out serum in second day, blood clot is collected serum again with the centrifugal 15min of 4000r/min, and-20 ℃ of preservations are standby.

Expand method by two-way fine jade, with 100mL 0.8%NaCl solution dissolving 1g agarose, behind 115 ℃ of autoclaving 10min, pour in the double dish, make the gel slab behind the 4mm.Punch at gel slab with card punch, serum (1: 2,1: 4,1: 8,1: 16,1: 32) and the negative rabbit anteserum of doubling dilution are dripped respectively in quincunx outer perimeter holes, interstitial hole drips reorganization UL51 albumen or the DPV of purifying, and 37 ℃ of wet boxes are hatched 24～48h, observes fine jade and expands the result.The result shows, apparent fine jade all occurred in 1: 2,1: 4,1: 8,1: 16 and 1: 32 to expand precipitation line, and negative rabbit anteserum do not have precipitation line (Fig. 4), and this shows reorganization UL51 albumen, has preferably antigenicity and can react with DPV.

1.2.2 the purifying of the anti-reorganization of rabbit UL51 protein antibodies IgG

A, caprylic acid-ammonium sulfate method are slightly carried the anti-reorganization of rabbit UL51 protein antibodies IgG: slightly carry the anti-reorganization of rabbit UL51 protein antibodies IgG, step is as follows: (1) gets the 0.06mol/L pH4.8 acetate buffer of the 10mL serum adding equivalent of separation, mixing; (2) dropwise add caprylic acid 75 μ g/mL serum under the stirring at room, stirring at room 30min, 4 ℃ leave standstill＞3h, make its abundant post precipitation, the centrifugal 30min of 13000r/min; (3) get supernatant and filter with filter paper, add the PBS of the 10mmol/LpH7.4 of 1/10 volume in the filtrate, regulate pH=7.4 with 2mol/L NaOH; (4) slowly add saturated ammonium sulfate 0.277g/mL in the liquid under the ice bath and make its final concentration reach 45%, stir 30min, put 4 ℃ to leave standstill＞3h or spend the night, 13000r/min is centrifugal, and 30min abandons supernatant; (4) add PBS in 1: 4 ratio in precipitation, fully piping and druming is fully dissolved it; (5) lysate is packed in the bag filter, 4 ℃ down with 10mmol/L pH7.4PBS dialysis, every 6h changes liquid once, until Nessler's reagent detect do not have precipitation in the dislysate and produce till.

B, the anti-reorganization of High Q post anion-exchange chromatography purified rabbit UL51 protein antibodies IgG: antibody purification IgG, step is as follows: (1) sample pre-treatments: will slightly carry IgG and remove impurity with 0.45 μ m membrane filtration, carrying out dialysis equilibrium with buffer A (pH8.0,20mmol/LTris-HCl damping fluid) under 4 ℃ of conditions spends the night; (2) chromatographic column pre-treatment: through pre-treatments such as 0.5mol/LNaOH, washing, 1.0mol/LNaCl, washings, and with buffer A balance HighQ chromatographic column; (3) go up sample: that gets 5mL slightly carries the IgG sample; (4) clean: with 50mL buffer A balance and wash unconjugated protein and impurity; (5) wash-out: (1.0mol/L NaCl) carries out linear gradient ionic strength wash-out with buffer B, flow velocity 0.5mL/min, and elution time is 200min, the fraction collection elution fraction.

C, IgG antibody purity and concentration determination: after the eluting peak sample preparation of collecting, carry out the SDS-PAGE electrophoresis, measure the purity of IgG antibody, and measure the ultimate density of IgG antibody with the Bradford method, and with its dilution for 2mg/mL, standby in-20 ℃ of preservations after the packing.

1.3 the preparation of mouse-anti reorganization UL51 protein antibodies IgG and purifying (repeat three these tests, each 1 week at interval is to prepare the antibody of three different batches)

Specifically comprise (1) immunogenic preparation: with the reorganization UL51 albumen of purifying, head exempts from equivalent complete Freund's adjuvant mixing and emulsifying abundant, and second and third exempts from the incomplete Freund's adjuvant mixing and emulsifying abundant, and the 4th exempts from not add adjuvant.(2) immune programme for children: select 20 of healthy Vista rats, head exempts from the previous day and takes a blood sample respectively, and separation of serum is made negative control.Preparation mouse-anti reorganization UL51 albumen hyper-immune serum immune programme for children such as table 1.Four exempt from back 14d plucks the eyeball bloodletting, collects blood.Place 1h, 4 ℃ of refrigerator overnight for 37 ℃.Separated out serum in second day, blood clot is collected serum again with the centrifugal 15min of 4000r/min, and-20 ℃ of preservations are standby.(3) tiring of two-way fine jade expansion method mensuration mouse-anti reorganization UL51 albumen hyper-immune serum is 1: 64.Other can be with reference to preparation and the purifying of the anti-reorganization of rabbit UL51 protein antibodies IgG.

The immune programme for children of table 1 mouse-anti reorganization UL51 protein polyclone antibody

The purifying of mouse-anti reorganization UL51 protein antibodies IgG: the purifying of polyclonal antibody is with reference to rabbit antivenom purification step, the mouse-anti reorganization UL51 protein antiserum that obtains is carried out purifying, measure the ultimate density of IgG antibody with the Bradford method, and its dilution is 2mg/mL, standby in-20 ℃ of preservations after the packing.Respectively the reorganization UL51 protein antibodies IgG of the mouse-anti behind the purifying and the anti-reorganization of rabbit UL51 protein antibodies IgG are carried out the SDS-PAGE evaluation, the result shows that this IgG antibody has very high purity.Measure the ultimate density of two kinds of IgG antibody with the Bradford method, and its dilution is 2mg/mL, standby in-20 ℃ of preservations after the packing.

In the present embodiment, elect the animal of two kinds of prepared reorganization UL51 protein antibodies as rat and rabbit, also can select other different genera animals for use.

2 experiment materials and sample preparation

2.1 detect bacterial strain, strain and sample

Normal DEF (DEF) nutrient solution, contain the strong poison of DPV the DEF nutrient solution, contain duck virus hepatitis virus (Duck virus hepatitis, DHV) duck embryo allantoic liquid, (Riemerella anatipestifer, the cloaca cotton swab of the negative duck of bacterial culture fluid RA), the bacterial culture fluid that contains duck Escherichia coli (E.coli), DPV and the sick duck cloaca cotton swab sample behind the strong poison of artificial challenge DPV provide by this laboratory to contain riemerella anatipestifer.

2.2 other main agents

Goat anti-rabbit igg antibody is available from the rich bio tech ltd that rises in Shanghai; Gold labeling antibody dilution, golden labeling antibody are preserved liquid, the plain film treating fluid of glass fibre, are provided by Shanghai gold mark bio tech ltd; The PB damping fluid (pH7.2) of other conventional reagent such as 0.01mol/L is configuration according to a conventional method all.

2.3 key instrument and articles for use

The three-dimensional specking instrument of YZ-3000, IN-5000 cutting machine, CM-4000 press mold machine, the plain film of water-absorption fiber, the plain film of glass fibre, white plastic backboard, plastic casing, drying agent, absorbent wool, nitrocellulose filter (NC film) etc. provide by Shanghai gold mark bio tech ltd.

2.4 the processing of testing sample

The processing of a cell culture fluid, duck embryo allantoic liquid or bacterial culture fluid

Fluid sample to be checked is carried out following cracking to be handled: add isopyknic lysate [2%TritonX-100/PBS (0.01mol/L in sample liquid to be checked, PH=7.2) solution] effect 5min (thermal agitation), 12000r/min, 4 ℃ of centrifugal 5min collect supernatant and act on detection.

The processing of b cloaca cotton swab sample

The disposal route of cloaca cotton swab is as follows: after wiping the sick duck cloaca of examination with aseptic cotton swab, put into the sterilization EP pipe that fills about 500 μ L physiological saline, with the EP pipe behind thermal agitation 1～3min on the vortex oscillator, with aseptic nipper with cotton swab on the EP tube wall repeatedly extruding back take out the method processing that remaining liquid press a.

3, the preparation of colloid gold test paper

3.1 the preparation of the anti-reorganization of the rabbit of colloid gold label UL51 protein antibodies (by Shanghai gold mark bio tech ltd preparation)

The preparation of colloid gold particle: use trisodium citrate reduction method, get 1% chlorauric acid solution 1mL, add the chlorauric acid solution that the 99mL ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the chlorauric acid solution that the disposable rapid adding of 1% trisodium citrate 1.6mL boils, continue to be heated to solution and transfer the black-and-blue shiny red that finally becomes to by faint yellow, continue heating 5min behind the colour stable, the room temperature cooling replenishes dehydration to original volume.

The preparation of colloid gold label antibody: need to determine the anti-reorganization of the rabbit the suitableeest stable quantity of UL51 protein antibodies and the suitableeest mark PH of mark, carry out mark.The immune colloid gold compound that obtains is abandoned supernatant through the ultracentrifugation purifying, with preserving the immune colloid gold compound that the outstanding loose red deposit of liquid is preliminary purification.Compare survey OD to preserve liquid _530nmValue, 4 ℃ keep in Dark Place.

3.2 the assembling of colloid gold test paper

Carry out the assembling of ICS according to conventional method, concrete steps are as follows: the PB damping fluid (pH 7.2) that (1) uses 0.01mol/L respectively with mouse-anti UL51 recombinant protein IgG antibody and goat anti-rabbit igg antibody is suitably after the dilution, to dilute mouse-anti UL51 recombinant protein IgG antibody and the goat anti-rabbit igg antibody of getting well with the three-dimensional specking instrument of XYZ-3000 and distinguish specking on the NC film, form detection line (test line, the T line) and nature controlling line (control line, the C line), T line and C line are at a distance of 0.7cm, back gauge apart from the NC film is respectively 0.9cm, after putting 37 ℃ of dry 2h, 4 ℃ of sealings are preserved standby.(2) the plain film of glass fibre is dipped in the anti-reorganization of the rabbit UL51 protein antibodies of the colloid gold label that suitably dilutes with golden labeling antibody dilution work, again it is dipped in the plain film treating fluid of glass fibre with the sealing nonspecific binding site, 37 ℃ of dried overnight are made gold mark pad.(3) respectively NC film, sample pad (absorbent wool), gold mark pad (the plain film of glass fibre), absorption pad (the plain film of water-absorption fiber) are bonded on the white plastic plate successively, be assembled into the detection test paper plate, and cut into the wide test strips of 0.4cm with the LN-5000 cutting machine, in its plastic casing of packing into, pack, built-in drying agent, 4 ℃ of preservations.

3.3ICS ratio juris and result judge

Detect the ICS ratio juris of DPV antigen: mouse-anti is recombinated the IgG antibody sandwich of UL51 albumen in the T line, and the goat anti-rabbit igg antibody bag is by in the C line, and the anti-reorganization of colloid gold label rabbit UL51 protein I gG antibody solidifies in the gold mark and fills up; When the DPV liquid in the sample is flowed through gold mark pad when going up the anti-reorganization of the rabbit UL51 protein I gG antibody of the golden mark that solidifies, with its in conjunction with forming " the anti-reorganization of the rabbit of DPVUL51 albumen-colloid gold label UL51 protein I gG antibody " compound, this compound continues to flow, mouse-anti reorganization UL51 protein I gG antibody on the T line is combined, form " the anti-reorganization of the rabbit UL51 protein I gG antibody of mouse-anti reorganization UL51 protein I gG antibody-DPV UL51 albumen-colloid gold label " compound, colloid gold particle enrichment and present redness on the T line, and the intensity that the T line takes on a red color is directly proportional with the content of DPV UL51 albumen; The anti-reorganization of the rabbit of unconjugated colloid gold label UL51 protein I gG antibody continues to flow forward, form " the anti-reorganization of the rabbit of goat anti-rabbit igg antibody-colloid gold label UL51 protein I gG antibody " compound with the sheep Hangzhoupro rabbit igg antibody that is fixed on the C line, colloid gold particle enrichment and present redness on the C line.Therefore, after on the sample pad of the test strips of horizontal positioned, dripping the sample liquid to be checked of about 100 μ L, observing response result in 15min: if C line, two red lines of T line occur, positive; If it is only at the C line red line is arranged, negative; Occur if only a red line is arranged or do not have any red line at the T line, then invalidate the test.

3.4 the optimization of test strips optimum test condition

Following condition makes up one by one, determines the optimum test condition of ICS method:

Bag the determining by concentration of the mouse-anti of purifying reorganization UL51 protein I gG antibody: the mouse-anti UL51 protein I gG antibody of recombinating is made A with the PBS damping fluid (pH 7.2) of 0.01mol/L: after not diluting (2mg/mL), B:2 and doubly diluting (1mg/mL), C:4 and doubly dilute (0.5mg/mL), D:8 and doubly dilute (0.25mg/mL), with the three-dimensional specking instrument of XYZ-3000 with its specking on the NC film, form the T line.The test paper of preparation under the constant situation of other condition, according to reaction result, is determined to reach the dilutability of the suitableeest mouse-anti reorganization UL51 protein I gG antibody of test strips susceptibility requirement, be working concentration.Multiple in the present embodiment dilutes all by volume, and multiple dilutes.

The bag of goat anti-rabbit igg antibody is determined by concentration: goat anti-rabbit igg antibody is made A with the PBS damping fluid (pH7.2) of 0.01mol/L: after not diluting (4mg/mL), B:2 and doubly diluting (2mg/mL), C:4 and doubly dilute (1mg/mL), D:8 and doubly dilute (0.5mg/mL), with the three-dimensional specking instrument of XYZ-3000 with its specking on the NC film, form the C line.The test paper of preparation under the constant situation of other condition, according to reaction result, is determined to reach the dilutability of the suitableeest goat anti-rabbit igg antibody of test strips susceptibility requirement, be working concentration.

Dilution the determining of the anti-reorganization of the rabbit of colloid gold label UL51 protein I gG antibody: the anti-reorganization of the rabbit of colloid gold label UL51 protein I gG antibody is made A: do not dilute (2mg/mL), B:2 doubly dilutes (1mg/mL), C:4 doubly dilutes (0.5mg/mL), after D:8 doubly dilutes (0.25mg/mL), be dipped in respectively in the dilution of variable concentrations with the plain film of onesize glass fibre, with the test paper of preparation under the constant situation of other condition, according to reaction result, determine to reach the dilutability of the anti-reorganization of rabbit UL51 protein I gG antibody of the suitableeest colloid gold label of test strips susceptibility requirement, be working concentration.

Through optimizing and screening, top condition based on the ICS of anti-reorganization UL51 protein antibodies is: the mouse-anti that (1) will dilute good purifying with the three-dimensional specking instrument of XYZ-3000 recombinate UL51 protein I gG and goat anti-rabbit igg antibody respectively with 1mg/mL and 1mg/mL specking on the NC film, form T line and C line, after putting 37 ℃ of dry 2h, 4 ℃ of sealings are preserved standby.(2) the plain film of glass fibre is dipped in the anti-reorganization of the rabbit UL51 protein I gG antibody with the colloid gold label of 2mg/mL, it is dipped in the plain film treating fluid of glass fibre with the sealing nonspecific binding site again, 37 ℃ of dried overnight are made the gold mark and are filled up.(3) respectively NC film, sample pad (absorbent wool), gold mark pad (the plain film of glass fibre), absorption pad (the plain film of water-absorption fiber) are bonded on the white plastic backboard successively, be assembled into the detection test paper plate, and cut into the wide test strips of 0.4cm with the LN-5000 cutting machine, in its plastic casing of packing into, pack, built-in drying agent, 4 ℃ of preservations see Fig. 5 for details.

The utilization of 4 colloid gold test papers

4.1 sensitivity tests

The cell culture fluid that will contain DPV did 1: 10,1: 20,1: 40,1: 80,1: 160 and 1: 320 doubling dilution after, detect with the ICS method of above-mentioned foundation, determine its detectable minimum extension rate.

When the result is diluted to 1: 80 when the cell culture fluid that contains DPV, visible two red stripes clearly still; When the cell culture fluid that contains DPV was diluted to 1: 160, only red stripes (Fig. 6) clearly appearred in visible C line.When this virus liquid was diluted to 1: 80 times, when concentration was about 1.25 μ g/mL, test strips presented weak positive reaction, owing to added 100 μ L sample to be checked in each well, so be 0.125 μ g with the detected DPV virus protein minimum content of ICS method energy.

4.2 specificity test

ICS method with above-mentioned foundation detects normal DEF nutrient solution respectively, contain the DEF nutrient solution of DPV, contain disease DHV the duck embryo allantoic liquid, contain the bacterial culture fluid of RA and contain E.coli bacterial culture fluid, observations.

The result shows visible two red stripes clearly of the DEF nutrient solution only contain DPV, and a red stripes clearly all only appears in other sample liquid at C line place.Show that the method has good specificity.

4.3 replica test

(1) criticize in repeatable test: detect normal DEF nutrient solution (1 part) respectively and contain the DEF nutrient solution (1 part) of DPV with the colloidal gold immuno-chromatography test paper strip method of above-mentioned foundation, do 3 repetitions, observations; (2) criticize between repeatable test: detect normal DEF nutrient solution (1 part) respectively and contain DPV malicious DEF nutrient solution (1 part) by force, observations with the test strips of 3 different batches preparations.

(1) criticizing interior repeatable test findings shows: 3 duplicate detection results by the described normal DEF nutrient solution of treated mistake and the DEF nutrient solution that contains DPV are all consistent; (2) repeatable test findings shows between criticizing: the test strips with 3 different batches preparations detects the normal DEF nutrient solution of handling respectively and contains the strong malicious DEF nutrient solution of DPV, and the result who obtains is equal unanimity also.The ICS method that shows foundation have in good batch and batch between repeatability.

4.4 stability test

Place 4 ℃, 25 ℃ and 37 ℃ respectively after will the test strips dry sealing, and detect normal DEF nutrient solution (1 part) and contain the DEF nutrient solution (1 part) of DPV, observations in March and June respectively.

After the dry sealing of test strips, place for 3～June at 4 ℃ and 25 ℃ respectively, can detect stronger positive findings; Place for 3～June at 37 ℃, still can detect stronger positive findings.Show that this test strips can preserve half a year 4 ℃ or 25 ℃ at least.

4.5 the detection to duck cloaca cotton swab sample

To 10 parts of cloaca cotton swab samples that do not infect the strong malicious duck of DPV and 10 parts of sick ducks of the strong malicious artificial challenge of DPV, after pressing the processing of 2.4b method, respectively with present embodiment based on the ICS method of anti-reorganization UL51 protein antibodies, detect based on AC-ELISA method and the conventional PCR method of anti-reorganization UL51 protein antibodies, and to testing result relatively.

Testing result is as shown in table 2: (1) 10 part of cloaca cotton swab test result of samples that does not infect the strong malicious duck of DPV: the negative rate unanimity of three kinds of methods all is 100%; The cloaca cotton swab test result of samples of the sick ducks of the strong malicious artificial challenge of (2) 10 parts of DPV: the positive rate that detects based on the AC-ELISA method of anti-reorganization UL51 protein antibodies is 100% (10/10), the positive rate that detects based on the ICS method of anti-reorganization UL51 protein antibodies is 90% (9/10), and both coincidence rates are 90% (9/10); Detecting with the coincidence rate of advising PCR method based on anti-AC-ELISA method of recombinating the UL51 protein antibodies is 100% (10/10); Be 90% (9/10) based on the ICS method of anti-reorganization UL51 protein antibodies and the coincidence rate of conventional PCR method.Above result shows that antigen capture ELISA method and conventional PCR method based on anti-reorganization UL51 protein antibodies that this research is set up have 100% coincidence rate, and are 90% based on the ICS method of anti-reorganization UL51 protein antibodies and the coincidence rate of above 2 kinds of methods.

3 kinds of comparisons that detect the DPV method of table 2

5 discuss

The ICS method of setting up in the present embodiment based on anti-reorganization UL51 protein antibodies has been used the principle of work of double antibody sandwich method, and double antibody sandwich method is the conventional method that detects antigen, has stronger specificity, is very suitable for checking various big molecular antigens.2 kinds of antibody in the classic method prepare with virus immunity different genera animal, and 2 kinds of antibody in this test then prepare with reorganization UL51 protein immunization different genera animal.

The disposal route of sample discharges from cell for DPV UL51 albumen and plays key effect.Usually the method for handling sample has multigelation method, chloroform give usage, ultrasonic treatment method, add lysate makes usage etc.Used several different methods to handle sample in preliminary experiment, testing result shown and added lysate to make the effect of usage best, therefore selected for use to add isopyknic lysate in the sample liquid to be checked and handle various samples to be checked.

Colloid gold test paper based on anti-reorganization UL51 protein antibodies has adopted the double antibodies sandwich principle, namely two kinds of anti-reorganization UL51 protein antibodies are used to bag respectively by NC film and mark collaurum, if contain DPV UL51 albumen in the testing sample, sample moves forward by the chromatography effect, arrive golden cursor position and the anti-reorganization of golden mark rabbit UL51 protein I gG antibody generation immune response, form " the anti-reorganization of the rabbit UL51 protein I gG antibody of DPV UL51 albumen-colloid gold label " compound; This compound continues to flow, IgG antibody generation immune response with mouse-anti on detection line reorganization UL51 albumen, form " the IgG antibody of the anti-reorganization of the rabbit UL51 albumen of the IgG antibody-DPV UL51 albumen-colloid gold label of mouse-anti reorganization UL51 albumen " compound, colloid gold particle enrichment and present redness on the T line, and the intensity that the T line takes on a red color is directly proportional with the content of DPV UL51 albumen; The anti-reorganization of the rabbit of unnecessary golden mark UL51 protein I gG antibody continues swimming to the C line, with goat-anti rabbit polyclonal antibody generation immune response, collaurum is developed the color at the C line.

In addition, determining of label working concentration is to guarantee test strips sensitivity and specific key factor, for guaranteeing the accuracy of etiologic diagnosis, needs strict control gold mark pad to go up the label concentration of antibody on immune colloid gold and the NC film.Excessive concentration can make NC film background deepen on the one hand, makes to detect to be with the colour developing result unintelligible, and non-specific responding is strengthened, and influences the specificity of qualitative results; Though concentration is crossed the low non-specific responding that reduced, reduced the sensitivity of reaction simultaneously again.In this test, through optimizing and screening, we have determined that the best effort concentration of mouse-anti reorganization UL51 protein I gG antibody, goat anti-rabbit igg antibody and the anti-reorganization of colloid gold label rabbit UL51 protein I gG antibody in the ICS method is respectively 1mg/mL, 1mg/mL and 2mg/mL.

When this test is done 1: 80 times of dilution with DPV virus liquid, still be positive, proved that this test paper has higher susceptibility, reason may have following several aspect: NC film, the glass fiber filter paper that (1) this test is selected for use has that the protein adsorption capacity is big, hydrophilicity good, need not advantages such as pre-service, therefore, although the package amount of antigen seldom, the antigen concentration on the NC film detection line is higher, makes the ICS method have higher susceptibility.(2) characteristics of the immune response of ICS method collection and chromatography, the gold mark rabbit anti-reorganization UL51 protein I gG antibody of DPV UL51 antigen in the testing sample on gold mark pad is combined, and forms " the anti-reorganization of the rabbit UL51 protein I gG antibody of DPV UL51 albumen-colloid gold label " compound; Continue by the NC film through the chromatography effect again, solid phase antigen is combined on the NC film, because all samples is all through the reaction of the plain film generation of fibre strip continuation, gold grain continues to assemble and the T line is deepened gradually, be actually antibody to be measured has been played an inspissation, thereby improved the susceptibility of immunochromatographic method.In addition, in the process of setting up the ICS method, we have selected to contain the duck embryo allantoic liquid of DHV, the bacterial culture fluid that contains the bacterial culture fluid of RA and contain E.coli is done the specificity experiment, and the result shows this ICS method and the equal no cross reaction of these several cause of diseases, has specificity preferably; In batch and batch between repeatable test confirmed that this method has good repeatability; Stability test shows that this test strips preserves half a year 4 ℃ or 25 ℃, still can be used for sample detection.

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 not depart from the spirit and scope of the present invention that appended claims limits.

Claims

1. based on the colloid gold test paper of anti-reorganization UL51 protein antibodies, formed by base plate, nitrocellulose film, sample pad, gold mark pad and absorption pad, p-wire and control line are arranged on the nitrocellulose film, it is characterized in that: the p-wire on the described nitrocellulose film is formed by the anti-reorganization of the A UL51 protein antibodies IgG bag that concentration equals 1mg/mL, and control line equals the anti-B IgG bag of 1mg/mL C by concentration and formed; Described gold mark pad is formed by the anti-reorganization of the B UL51 protein antibodies IgG bag that colloid gold label concentration equals 2mg/mL; The anti-reorganization of described A UL51 protein antibodies IgG is mouse-anti reorganization UL51 protein antibodies IgG, and the anti-reorganization of described B UL51 protein antibodies IgG is the anti-reorganization of rabbit UL51 protein antibodies IgG, and the anti-B IgG of described C is goat anti-rabbit igg.

2. the method for making of the described colloid gold test paper based on anti-reorganization UL51 protein antibodies of claim 1, it is characterized in that: concrete steps are:

The spray of p-wire and control line on a nitrocellulose film: the anti-reorganization of the A UL51 protein antibodies IgG specking that concentration is equaled 1mg/mL forms p-wire on the nitrocellulose film, concentration is equaled the anti-B IgG of 1mg/mLC specking form control line on the nitrocellulose film, dry back low temperature seal is preserved standby;

The preparation of the golden mark pad of b: the plain film of glass fibre is dipped in colloid gold label concentration equals among the anti-reorganization of the B UL51 protein antibodies IgG of 2mg/mL, again this film is dipped in the plain film treating fluid of glass fibre and seals nonspecific binding site, get golden mark pad after the drying;

3. the method for making of the colloid gold test paper based on anti-reorganization UL51 protein antibodies according to claim 2, it is characterized in that: the distance of described p-wire and described control line is 0.7 centimetre, and described p-wire and described control line are respectively 0.9 centimetre apart from the back gauge of nitrocellulose film.

4. the described application of colloid gold test paper in preparation duck plague virus antigen detection test paper based on anti-reorganization UL51 protein antibodies of claim 1.