CN102680699B - ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection - Google Patents
ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection Download PDFInfo
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Abstract
The invention discloses an ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection. The method is characterized by taking an FAVI 100K recombinant protein as the envelop antigen, chicken serum as the detection sample and horse radish peroxidase labeled goat anti-chicken IgG as the enzyme-labeled antibody to detect the antibody generated through FAVI infection. The method is strong in specificity, short in time and low in cost, is simple to operate, can be used for mass detection, can effectively get rid of interference of immunity of FAVI inactivated vaccines and can specifically detect FAVI infection, thus distinguishing FAVI infected animals from FAVI inactivated vaccine inoculated animals and providing a diagnostic tool with actual value for eliminating FAVI in China.
Description
Technical field
The invention belongs to technical field of biological, particularly a kind of indirect ELISA method that utilizes 100K recombinant protein to differentiate animal with the animal through immunity inoculation I group I fowl adenovirus inactivated vaccine of the infection of I group I fowl adenovirus.
Background technology
I group I fowl adenovirus (Fowl adenovirus group I, hereinafter to be referred as FAVI) be attributed to Adenoviridae Aviadenovirus, there is common group antigen, based on hexon gene order, be divided into 5 different kinds (A-E), based on neutralization test result, be subdivided into 12 serotypes.FAVI is ubiquity worldwide, and host is more in chicken, duck, goose body, can from the poultry of healthy and morbidity, isolate.This disease both can, through excreta horizontal transmission, also can, through ovum vertical transmission, be polluted chicken embryo.In recent years, the diseases such as the inclusion body hepatitis, hydropericardium hepatitis syndrome and the gizzard erosion that are caused by FAVI, the cultivation of the production to broiler chicken and kind chicken brings certain threat, thereby causes prevention and the control of aquaculture to this disease.What FAVI was more is copy in vivo and do not fall ill, and after this virus of zoogenetic infection, is subclinical infection, with other cause of disease actings in conjunction in poultry.After the inactivated vaccine immunity of FAVI, if set up the indirect ELISA method of a kind of FAVI of differentiation immune animal and infection animal, this disease of prevention and control, purification and elimination is had to vital effect.
At present, the serological method of detection FAVI mainly contains agar immunity diffusion, neutralization test, immunofluorescence, counter immunoelectrophoresis, indirect hemagglutination (IHA) and Southern hybridization etc.These methods are consuming time, effort, are not suitable for large batch of detection blood serum sample, and the antigen using mostly is totivirus, the loose malicious danger of existence.
100K is the non-structural protein of FAVI, that FAVI infects in the later stage the rich in protein of content, can be combined with up-to-date synthetic main structural proteins hexon, make the latter occur curling and be folded into homotrimer, can make hexon assemble from cytoplasm endoplasmic reticulum transporte to cells core simultaneously.100K can also be combined with adenovirus mRNA in late period, to promote the synthetic of virus protein, suppresses the synthetic of host protein simultaneously.
The ultimate principle that the indirect ELISA differential diagnostic method of 100K recombinant protein of the present invention is set up is that viral non-structural protein is only expressed in virus replication breeding, stimulates body to produce non-structural protein antibody.The inactivated vaccine adopting in the anti-system of epidemic disease at present, most non-structural proteins in preparation process, are destroyed, therefore use inactivated vaccine immune animal, in animal body, just only have structural proteins antibody in theory and the non-structural protein antibody that rarely even can't detect by existing method.And after the wild poison of zoogenetic infection, in the process that virus copies in vivo, breeds, just have the expression of non-structural protein, therefore can utilize the existence of non-structural protein antibody whether to distinguish infection animal and inactivated vaccine immune animal.Large quantity research shows, the ELISA method of utilizing viral non-structural protein to set up can be distinguished and infect the antibody producing with inactivated vaccine immunity, as Raue R etc. utilizes bovine viral diarrhoea NS gene to set up commercialization ELISA kit, infect and the immune antibody producing of inactivated vaccine to distinguish; China Taiwan's scholars Shu PY etc. utilizes encephalitis B non-structural protein NS 1 to set up the indirect ELISA method of distinguishing infection and immunity antibody; Bruderer U etc. utilizes recombinant protein 3ABC to set up the ELISA method of the antibody of distinguishing aftosa natural infection and vaccine immunity; Makkay AM etc. utilizes ewcastle disease NP gene to set up the ELISA method of distinguishing natural infection and HN gene subunit vaccine chicken antibody; Laviada MD etc. build african horse sickness NS3 gene recombination bacterium, distinguish the method for natural infection and inactivated vaccine immunity.But, there is no at present the research that utilizes 100K recombinant protein to differentiate the ELISA detection method of I group I fowl adenovirus infection and report.
Summary of the invention
The present invention be directed to the deficiency in above-mentioned field, provide a kind of 100K of utilization recombinant protein to differentiate the ELISA detection method that I group I fowl adenovirus infects, the method high specificity, simple to operate, consuming time short, cost is low, can mass detection sample.
The present invention is achieved by the following technical solutions:
Differentiating the ELISA detection method that I group I fowl adenovirus infects, is taking FAVI100K recombinant protein as envelope antigen, and chicken serum is for detecting sample, and the goat-anti chicken IgG of horseradish peroxidase-labeled is that ELIAS secondary antibody detects the antibody that the infection of I group I fowl adenovirus produces.
Described envelope antigen is to prepare through following steps:
(1) prokaryotic expression of FAVI100K recombinant protein: taking the DNA of FAVI CELO strain as template, with Auele Specific Primer pcr amplification 100K (GenBank:U46933:23671-24732nt) genes of interest, genes of interest is cloned in pGEX-4T-1 prokaryotic expression carrier, transforms bacillus coli DH 5 alpha competent cell.Extract clone according to amicillin resistance, the plasmid DNA of rapid extraction transformant, double digestion qualification, obtains the fragment consistent with object fragment molecular weight, is judged to positive findings, called after 100K-pGEX.Positive plasmid send the order-checking of the precious biotech firm in Dalian, result shows that FAVI100K recombinant protein genes of interest sequence is correctly connected into pGEX-4T-1, size is 1062bp, reckoning protein molecular weight is 38.9ku, the molecular mass of the glutathione sulfydryl transferase (GST) that pGEX-4T-1 carrier itself is expressed is 25.6ku, therefore, the molecular weight of FAVI100K recombinant protein is 64.5ku, and the primer of 100K Gene Partial sequence amplification is as follows:
Upstream primer: 5 '-CCG
gAATTCtCATCGAAAATGGCAGACAAGAT-3 ', inserts EcoR I restriction enzyme site;
Downstream primer: 5 '-ATTT
gCGGCCGCcCCGAGACGCCATTTAGGTA-3 ', inserts Not I restriction enzyme site;
(2) FAVI100K recombinant protein is identified: the recombinant plasmid 100K-pGEX of genes of interest is transformed to bacillus coli DH 5 alpha, after IPTG induction, mycoprotein shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compared with empty thalline, there is a dense newly-increased protein band dying at about 64.5ku place.Through western-blot qualification, can react with FAVI positive serum, and empty thalline does not react, and shows that FAVI100K recombinant protein has stronger reactionogenicity;
(3) the affinity chromatography method through glutathione agarose resin adsorption, washing, wash-out by the FAVI100K recombinant protein after qualification, the 100K-pGEX recombinant protein of acquisition purifying, i.e. envelope antigen.
Described chicken serum comprises FAVI negative serum, experimental infection serum and inactivated vaccine immune serum.
Differentiate the ELISA detection method that I group I fowl adenovirus infects, comprise the steps:
(1) coated: envelope antigen is diluted to 1～20 μ g/mL with coating buffer, coated 96 hole ELISA Plate, every hole 100 μ L, after 37 DEG C of incubation 1h, are placed in 4 DEG C of 12～16h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds confining liquid 200 μ L, hatches for 37 DEG C, sealing 1h, and PBST washes plate 3 times, pats dry;
(3) be combined with serum: add detection sample, 100 μ L/ holes, establish negative standard items and positive criteria product for contrast, hatch after 1h in 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: the goat-anti chicken IgG dilution of horseradish peroxidase-labeled is doubly diluted with 1:1000～1:8000, and every hole adds 100 μ L, is placed in 37 DEG C and hatches after 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L substrate nitrite ions, in 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L stop buffers, cessation reaction;
(7) result is judged: read 450nm wavelength place light absorption value, i.e. OD450 value in microplate reader;
Calculate OD450 mean value X and the standard deviation SD of FAVI negative serum, according to principle of statistics, the X+3SD of FAVI negative serum is as yin and yang attribute critical value, FAVI negative serum sample at least wants 30 parts just to reach above statistical requirements, if detect sample OD450 value > yin and yang attribute critical value, be judged to the positive; If sample OD450 value < yin and yang attribute critical value, is judged to feminine gender.
Wherein, described coating buffer is carbonate buffer solution: Na
2cO
31.59g, NaHCO
32.93g, adding distil water is settled to 1000mL, adjust pH to 9.6;
Described PBST is the phosphate buffer that contains 0.05% Tween-20: NaCl8.0g, KCl0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o2.9g, Tween-20 500 μ L, adding distil water is settled to 1000mL, adjust pH to 7.4;
Described confining liquid is 5% skimmed milk, and 5g skimmed milk is settled to 100mL with PBST;
Described dilution is 1%BSA, and 1g bovine serum albumin(BSA) BSA is settled to 100mL with PBST;
Described substrate nitrite ion is: TMB damping fluid 10mL, TMB solution 0.5mL, H
2o
232 μ L;
Described stop buffer is 2M H
2sO
4, the 21.7mL concentrated sulphuric acid slowly adds in 178.3mL distilled water.
Described negative standard items are for hatching the serum of produced 2～5 Japanese instar chicklings with SPF chicken embryo.
Described positive criteria product comprise inactivated vaccine positive criteria product and experimental infection positive criteria product; Inactivated vaccine positive criteria product are the SPF chicken serum after twice subcutaneous inoculation of I group I fowl adenovirus inactivated vaccine; Experimental infection positive criteria product are the SPF chicken serum after twice of mode collunarium, the eye droppings infecting through I group I fowl adenovirus simulating nature.
The invention has the beneficial effects as follows:
1. the present invention chooses the 100K Gene Partial sequence of FAVI CELO strain, pass through clonal expression, using the 100K recombinant protein of prokaryotic expression as antidiastole antigen, adopt indirect ELISA pattern, develop FAVI non-structural protein antibody assay kit, provide a kind of diagnostic tool that has actual value for China eliminates FAVI.This method is simple to operate, and consuming time short, cost is low, can mass detection.
2. the present invention chooses the partial sequence of the 100K recombinant protein that FAVI antigenicity is stronger, by analyzing, selects epitope and the good region of water wettability wherein, adopts gene recombination technology, and prokaryotic expression, obtains a kind of new 100K recombinant protein after affinity chromatography.Both retain its antigenicity through transformation 100K recombinant protein, improved again the prokaryotic expression amount of 100K recombinant protein.This recombinant protein not only has high specific as antigen, and has high-purity and high stability, is extracting after purifying, and wrapper sheet is as the important component in detection method.Compared with totivirus antigen, this recombinant antigen can scale, standardized production, safer, economical.
3. the present invention is directed to production of vaccine technique and the animal immune situation of the actual use in China's animal and veterinary industry field, determined criterion.FAVI, after inactivated vaccine immunity inoculation, due to non-structural protein destroyed in the production run of vaccine, does not produce the antibody of non-structural protein, but in FAVI course of infection, produces the antibody of non-structural protein.Therefore, the present invention utilizes FAVI100K non-structural protein, build 100K recombinant protein as envelope antigen, set up indirect ELISA antibody detection method, can detect that FAVI infects the antibody producing, and can not detect the antibody of inactivated vaccine immunity, thereby can effectively distinguish I group I fowl adenovirus infection animal and I group I fowl adenovirus inactivated vaccine immune animal.
Brief description of the drawings
Fig. 1: the SDS-PAGE of 100K recombinant protein analyzes
M. protein molecule standard; The empty thalline of 1.pGEX-4T-1; 2.100K-pGEX not induction; 3.100K-pGEX the expression product of induction.
Fig. 2: the western-blot of 100K recombinant protein analyzes
M. protein molecular weight standard; 1. empty map; 2.100K recombinant protein.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to the scope that embodiment represents.
Implement 1:
One, the clone of 100K gene
The extraction of 1.I group I fowl adenovirus DNA
Use day root biochemical technology blood/cell/tissue genome DNA extracting reagent kit, the allantoic fluid directly going down to posterity from the kind poison of the CELO virus purchased from China Veterinery Drug Inspection Office (CELOV), extract DNA, operation is undertaken by kit instructions, and concrete steps are as follows:
(1) get allantoic fluid 180 μ L, add 20 μ LGA, 20 μ L Proteinase Ks mix, 56 DEG C of 4h digestion.
(2) add 200 μ L damping fluid GB, fully put upside down and mix, place 10 minutes for 70 DEG C, solution strain is limpid, brief centrifugal to remove the globule of cap wall.
(3) add 200 μ L absolute ethyl alcohols, fully vibration mixes 15 seconds, now may occur flocculent deposit, brief centrifugal to remove the globule of cap wall.
(4) previous step gained solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column is put into collection tube), centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
(5) in adsorption column CB3, add 500 μ L damping fluid GD, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
(6) in adsorption column CB3, add 700 μ L rinsing liquid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
(7) in adsorption column CB3, add 500 μ L rinsing liquid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid.
(8) adsorption column CB3 is put back in collection tube, centrifugal 2 minutes of 12000rpm, outwells waste liquid.Adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(9) adsorption column CB3 is proceeded in a clean centrifuge tube, to the unsettled dropping 50 μ L elution buffer TE in middle part of adsorption film, room temperature is placed 2-5 minute, centrifugal 2 minutes of 12000rpm, solution is collected to centrifuge tube, preserve or carry out immediately pcr amplification for-20 DEG C.
2.PCR amplification
Taking the DNA of gained in above-mentioned steps 1 as template, use upstream primer
5 '-CCG
gAATTCtCATCGAAAATGGCAGACAAGAT-3 ' and downstream primer
5 '-ATTT
gCGGCCGCcCCGAGACGCCATTTAGGTA-3 ' carries out pcr amplification.
Reaction system 100 μ L, as follows: the PCR MasterMix50 μ L of Tian Gen biochemical corp, upstream primer (10uM) 2 μ L, downstream primer (10uM) 2 μ L, template DNA 2 μ L, aqua sterilisa 44 μ L.
After moment centrifugal mixing, the response procedures of pcr amplification is as follows: 94 DEG C of 5min denaturations, and 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 35 circulations, 72 DEG C of 8min extend.PCR product is through 1% agarose electrophoresis (100-120V), and amplified production fragment is consistent with the object clip size of expection, can be used for downstream tests.
3. the purifying of target DNA fragment
PCR product, after 1% agarose gel electrophoresis 50min, is cut to the Ago-Gel piece that contains target DNA and is placed in 1.5mL centrifuge tube under uviol lamp.
Reclaim kit instructions by the AxyPrep of AXYGEN company DNA gel and test, concrete steps are as follows:
(1) calculated for gel weight (recording in advance 1.5ml centrifuge tube weight), this weight is as a gel volume (as 100mg=100 μ L volume)
(2) add the Buffer DE-A of 3 gel volumes, after mixing, in 75 DEG C of heating, be interrupted and mix (every 2-3min), until gel piece melts (about 6-8min) completely
(3) add the Buffer DE-B of 0.5 Buffer DE-A volume, mix.
(4) draw the mixed liquor in step 3, transfer to DNA preparation pipe (2mL (providing in kit) centrifuge tube is provided), the centrifugal 1min of 12000 × g.Abandon filtrate.
(5) put back 2mL centrifuge tube by preparing pipe, add 500 μ L Buffer W1, the centrifugal 30s of 12000 × g, abandons filtrate.
(6) put back 2mL centrifuge tube by preparing pipe, add 700 μ L Buffer W2, the centrifugal 30s of 12000 × g, abandons filtrate.Wash a centrifugal 1min of 12000 × g with 700 μ L Buffer W2 again with same method.
(7) put back in 2mL centrifuge tube the centrifugal 1min of 12000 × g by preparing pipe.
(8) be placed in clean 1.5mL centrifuge tube by preparing pipe, add 30 μ L and remove centrifugal water preparing film central authorities, room temperature leaves standstill 1min.The centrifugal 1min eluted dna of 12000 × g.
4. target DNA fragment is connected with pGEX-4T-1 carrier
Target DNA and pGEX-4T-1 plasmid are all used EcoR I and Xho I double digestion, and the enzyme system of cutting is 20 μ L, as follows respectively: target DNA (0.5-1ug/ μ L) 1 μ L, EcoR I1 μ L, Xho I1 μ L, 10 × buffer2 μ L, dH
2o15 μ L; PGEX-4T-1 plasmid (0.5-1ug/ μ L) 1 μ L, EcoR I1 μ L, Xho I1 μ L, 10 × buffer2 μ L, dH
2o15 μ L.37 DEG C of digestion 3h.Both double digestion products are run after glue purification, with the precious biological DNA ligation kit connection in Dalian, pGEX-4T-1 plasmid vector DNA is mixed with into the DNA solution that volume is 5-10 μ L (TE solution or aqueous solution) with inserting DNA fragmentation, and the mole ratio of carrier DNA and insertion DNA is generally: 0.03pmol:0.1-0.3pmol.To the Solution I that adds equal-volume (5-20 μ L) in above-mentioned DNA solution, fully mix.16 DEG C are reacted 30 minutes.Reactant liquor can be directly used in bacterium and transform.
5. the preparation of competent cell
(1) take out and be kept at-70 DEG C of bacillus coli DH 5 alpha bacterial classifications, be inoculated on LB flat board, cultivate 16h left and right for 37 DEG C.
(2) picking colony, to LB overnight incubation, next day, is transferred to 1:100 ratio in the culture flask of 50mL LB, cultivates 2.5-3h left and right for 37 DEG C, monitors bacterium liquid OD value, in the time of OD600=0.35, stops cultivating.
(3) bacterium liquid 50mL is poured in centrifuge tube into ice bath 10min.
(4) 0 DEG C of centrifugal 8min of 3000 × g.
(5) by nutrient solution evacuation, be inverted centrifuge tube 1min on thieving paper, make last evacuation.
(6) bacterium liquid and CaCl
2add CaCl with 5:3 ratio
2, add 30mL CaCl
2(precooling in advance), to bacterial sediment, will precipitate resuspended.
(7) 0 DEG C of centrifugal 10min of 3000 × g, precipitation thalline.
(8) repeating step (5).
(9) with 2mL precooling CaCl
2the resuspended precipitation of solution, and add 20% glycerine, mix, be sub-packed in 1.5mL centrifuge tube with every pipe 200 μ L, place-70 DEG C of preservations.
6. the conversion of competent cell
(1) the competent cell 200 μ L of-70 DEG C of preservations of taking-up, add 10-20 μ L to connect product after thawing, softly mix ice bath 30min.
(2) be placed in 42 DEG C of water-bath heat stress 90s, take out ice bath heat shock 2-3min.
(3) add 800 μ L LB nutrient solutions, 37 DEG C of shaking tables are cultivated 45min, make cell recovery.
(4) with liquid-transfering gun, cultured bacterium liquid is beaten lentamente on plating medium surface, then used flat board under hand rolling, make the abundant spread plate of bacterium liquid.
(5), after drying up, be inverted in 37 DEG C of constant temperature ovens and cultivate.
7. choose spot
The single bacterium colony of toothpick picking from flat board at ultra-clean Biohazard Safety Equipment with sterilization, is inoculated in 1mL LB nutrient culture media (containing 50ug/mL ampicillin), and 37 DEG C about shaken cultivation 16h hour.
8. the extraction of plasmid and enzyme are cut qualification
Extract the plasmid of recombinant vector bacterium according to the little extraction reagent kit instructions of the plasmid of Tian Gen biochemical corp, step is as follows:
(1) get the bacterium liquid of 5mL incubated overnight, add in centrifuge tube, use conventional desk centrifuge, centrifugal 1 minute of 12000rpm absorbs supernatant as far as possible.
(2) add 250 μ L solution P1 to leaving in the centrifuge tube of bacterial sediment, bacterial precipitation thoroughly suspends.
(3) in centrifuge tube, add 250 μ L solution P2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.
(4) in centrifuge tube, add 350 μ L solution P3, leniently spin upside down 6-8 time immediately, fully mix, now occur white flocculent deposit.The centrifugal 10min of 12000rpm, now forms precipitation in centrifuge tube bottom.
(5) supernatant of previous step being collected is transferred in adsorption column CP3 with pipettor, notes trying not to be drawn onto precipitation.The centrifugal 30-60 of 12000rpm second, outwell the waste liquid in collection tube, adsorption column CP3 is put into collection tube.
(6) in adsorption column CP3, add 600 μ L rinsing liquid PW, the centrifugal 30-60 of 12000rpm second, outwell the waste liquid in collection tube, adsorption column CP3 is put into collection tube.
(7) adsorption column CP3 is put into collection tube, centrifugal 2 minutes of 12000rpm, object is that rinsing liquid remaining in adsorption column is removed.
(8) adsorption column CP3 is placed in to a clean centrifuge tube, drips 50-100 μ L elution buffer EB to the middle part of adsorption film, room temperature is placed 2 minutes, and 12000rpm collects plasmid solution in centrifuge tube for centrifugal 2 minutes.
The 100K-pGEX plasmid extracting with EcoR I and Xho I double digestion, 20 μ L systems are as follows: EcoR I1 μ L, Xho I1 μ L, 100K-pGEX plasmid 5 μ L, add aqua sterilisa to 20 μ L.37 DEG C of digestion 3h.Run enzyme through 1% agarose electrophoresis and cut qualification figure.
(9) order-checking of recombinant plasmid qualification: pcr amplification qualification, enzyme are cut to all positive clones of qualification, and incubated overnight amplification, serves the order-checking of Hai Yingjun company.Identify correct recombinant plasmid called after 100K-pGEX.
The nucleotide sequence of gained coding 100K fragment is as follows:
TCATCGAAAATGGCAGACAAGATTACCCGAGAGGAAAAAACCATAGCGACGCTGGACCTCGTGTTACGCGTGGTCGTCGATGCTGGTAACTGGGACGTGTTCTCGAAACGTTTGGTTCGCTACACACGCGAACAGTACGGAATCGAGCTGCCCGAAGATATCGGGGACTTACCGGACACATCTGAGGTCTCGAAAGTGCTGTTGAGTCATTTGGGGGAAGACAAGGCGGTACTGTCCGCGTACCGAATCGCGGAACTGACGCAACCTTCCGAAATGGACCGCGCTAAGGTCACAGAGGGAGGCCTGGCCGTACTTAACGCGAGTCGCGATGAAAGCGAAGCTCAGAACCCCTCGAACCCCGAACCCGAGAGCATCGAGAGCGACGCCGTAGAGGATCTCGGCGTTGCAGCAGAGAGCGACCCTAGCGATGACGAACCCGACCCAGAACCCGAGTATGACCATCGAGAGGCGGATCATGACTCTGATGCGGATAGCGGATACTATTCGGCAGATGGGGGACGACCTGGAACACCAGTGGACGAGGAGCCCCAGGACGATTCTCCCTCTTCCGAGGAGACCGCATCCACTGTCATCGAAGAAGCGCAGACTAGCGCTAGCAACGATTCTCATGACGACGACACTCACCGCGACGACGGCAGTGCTTCTGAAGAGGATCTCGAGCGGGACGCCCTCGTGGCCCCGGCCGATCCTTTTCCCAACTTGCGGAAGTGTTTCGAGCGCCAAGCCATGATGCTGACCGGGGCGTTAAAAGACGCCGCGGACACGGCTGATCCGCCAGAAACGCTCTCCGTCGACAGCGTGCAAAGGCAGCTCGAACGCTTCGTCTTTAACCCCGACCGCCGCGTGCCCGCCGAACACTTGGAGGTACGCTACAATTTCTACCCTCCTTTCCTCACCCCCAAGGCCATCGCGAGCTATCACATCTTTGCCGTCACCGCTTCCATCCCTCTAAGCTGCAAAGCCAACCGCAGCGGCAGCGACCTTCTAGCCAAAGCAAAAGAGAGCACTTTCTTCAAACGCTTACCTAAATGGCGTCTCGGG
Two, the abduction delivering of recombinant expression plasmid
To be seeded in the LB nutrient solution that 5mL contains ampicillin (50ug/mL) 37 DEG C of shaken overnight through sequential analysis correct recombinant bacterium 50 μ L.Next day, getting overnight culture is seeded to 1:100 in the LB nutrient solution that contains ampicillin (50ug/mL), 37 DEG C vibrate while being 0.55-0.65 to OD600, add IPTG to final concentration 1mmol/L, 37 DEG C of abduction delivering 5h, carry out SDS-PAGE and western-blot and analyze, detect expression product.Establish the bacterium liquid of the empty thalline of pGEX-4T-1 and not induction as negative control simultaneously.
Three, the detection of expression product
Prepare the instructions of SDS-PAGE glue by Bole company, prepare protein adhesive.By the centrifugal 5min of bacterium liquid 12000rpm after induction, with 1/10 PBS suspension bacterial sediment of original bacteria liquid, add 4 × SDS loading buffer, mix, 5min is boiled in water-bath, in the sample well loading of protein adhesive, 10 μ L/ holes, first use 90V voltage electrophoresis 25min, voltage is mentioned to 120V, continue electrophoresis to bromophenol blue and arrive gel bottom.With the dyeing of coomassie brilliant blue staining liquid and destainer decolouring, see accompanying drawing 1.Result shows, obtains good expression through the 100K recombinant protein of induction, occur special band of expression, and the 100K recombinant protein of the empty thalline of pGEX-4T-1 and not induction is without this band of expression at about 64.5ku place.
Four, a large amount of preparation and purifications of 100K recombinant protein
By 100K-pGEX expression product multigelation 3 times, ultrasonic treatment, the centrifugal 10min of 12000 × g, gets cleer and peaceful precipitation and carries out SDS-PAGE electrophoresis, determines that 100K-pGEX recombinant protein mainly expresses at supernatant and still precipitate.Utilize the method for affinity chromatography, first use glutathione agarose resin adsorption recombinant protein, more repeatedly clean several times with PBS, remove DNA, RNA and foreign protein, finally use reduced glutathione damping fluid 100K-pGEX recombinant protein wash-out.
Five, western-blot analyzes
The good 100K recombinant protein of purifying, after SDS-PAGE electrophoretic separation, with the dry instrument that turns of the Western-blot of Shanghai Ying Jun company, forwards protein band to nitrocellulose filter (NC film) upper, establishes the contrast of sky thalline simultaneously.37 DEG C of 1h of 5% skimmed milk sealing for NC film, with PBST washing 3 times, each 5min, the FAVI positive serum that adds 1:50 to dilute, hatches after 1h for 37 DEG C, again with PBST washing 3 times, add the horseradish peroxidase-labeled goat-anti chicken IgG of 1:500 dilution, hatch 1h for 37 DEG C, PBST washes after film 3 times, by DAB colour developing, observations.
Six, 100K indirect ELISA is set up
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, FAVI100K recombinant protein is diluted to working concentration 20ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 16h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:2000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, in table 1, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Table 1100K-ELISA detects the OD450 value of 50 parts of FAVI negative serums
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.Result shows as shown in table 2, table 3.
The OD450 value that table 2100K-ELISA test experience infection animal serum is 50 parts
Table 3100K-ELISA detects the OD450 value of 50 parts of inactivated vaccine immune serums
From table 2 and table 3,96% infection animal serum is positive, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 2:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 1ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 12h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:1000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 3:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 10ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 15h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:8000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 4:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 15ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 14h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:3000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 5:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 5ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 13h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:4000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 6:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 16ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 12h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:5000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 7:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 18ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 16h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:6000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Embodiment 8:
According to step 1 to the five preparation 100K recombinant protein in embodiment 1
Six, the foundation of 100K indirect ELISA detection method
100K recombinant protein after purifying is set up indirect ELISA method as antigen, and concrete step is as follows:
(1) coated: with coating buffer, the I group I fowl adenovirus 100K albumen by prokaryotic expression is diluted to working concentration 9ug/mL, adds 96 hole ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 1h, are placed in 4 DEG C of 13h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds the skimmed milk of 200 μ L5%, 37 DEG C of sealing 1h, PBST washes plate 3 times, pats dry;
(3) be combined with serum: add blood serum sample to be checked, 100 μ L/ holes, establish the positive and negative standard items for contrast, hatch 1h for 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: with the goat-anti chicken IgG of 1%BSA dilution horseradish peroxidase-labeled, to working concentration (1:7000 doubly dilutes), every hole adds 100 μ L, is placed in 37 DEG C and hatches 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L tmb substrate nitrite ions, 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L2M H
2sO
4cessation reaction, microplate reader is measured OD450 value.
(7) use the ELISA method of setting up to detect the negative serum of 50 parts of FAVI, under OD450nm wavelength, read absorbance value (OD450nm value) by microplate reader, calculate negative mean value (X) and standard deviation (SD), according to principle of statistics, the cut-off value that X+3SD is this detection method.The criterion that defines sample detection by the cut-off value of calculating is: in the time of sample OD450<0.345 to be checked, be judged to be feminine gender; In the time of sample OD450>0.345 to be checked, be judged to be the positive.
Seven, 100K indirect ELISA method is distinguished the animal of I group I fowl adenovirus infection and the animal of inactivated vaccine inoculation
Detect 100 parts of serum by 100K-ELISA method, comprised 50 parts of 50 parts of experimental infection animal blood serums and inactivated vaccine immune serums.96% infection animal serum is positive as a result, and 98% inactivated vaccine immune animal is negative, shows that the present invention can distinguish the animal of FAVI infection and the animal of inactivated vaccine inoculation.
Nucleotides sequence list
<110> Veterinary Institute of Guangxi Zhuang Autonomous Region
The ELISA detection method that <120> utilizes 100K albumen antidiastole I group I fowl adenovirus to infect
<210> 1
<211> 23
<212> DNA
<213> artificial sequence (upstream primer)
<400>
tcatcgaaaa tggcagacaa gat 23
<210> 2
<211> 20
<212> DNA
<213> artificial sequence (downstream primer)
<400>
cccgagacgc catttaggta 20
<210> 3
<211> 1062
<212> DNA
<213> I group I fowl adenovirus 100K
<400>
tcatcgaaaa tggcagacaa gattacccga gaggaaaaaa ccatagcgac gctggacctc 60
gtgttacgcg tggtcgtcga tgctggtaac tgggacgtgt tctcgaaacg tttggttcgc 120
tacacacgcg aacagtacgg aatcgagctg cccgaagata tcggggactt accggacaca 180
tctgaggtct cgaaagtgct gttgagtcat ttgggggaag acaaggcggt actgtccgcg 240
taccgaatcg cggaactgac gcaaccttcc gaaatggacc gcgctaaggt cacagaggga 300
ggcctggccg tacttaacgc gagtcgcgat gaaagcgaag ctcagaaccc ctcgaacccc 360
gaacccgaga gcatcgagag cgacgccgta gaggatctcg gcgttgcagc agagagcgac 420
cctagcgatg acgaacccga cccagaaccc gagtatgacc atcgagaggc ggatcatgac 480
tctgatgcgg atagcggata ctattcggca gatgggggac gacctggaac accagtggac 540
gaggagcccc aggacgattc tccctcttcc gaggagaccg catccactgt catcgaagaa 600
gcgcagacta gcgctagcaa cgattctcat gacgacgaca ctcaccgcga cgacggcagt 660
gcttctgaag aggatctcga gcgggacgcc ctcgtggccc cggccgatcc ttttcccaac 720
ttgcggaagt gtttcgagcg ccaagccatg atgctgaccg gggcgttaaa agacgccgcg 780
gacacggctg atccgccaga aacgctctcc gtcgacagcg tgcaaaggca gctcgaacgc 840
ttcgtcttta accccgaccg ccgcgtgccc gccgaacact tggaggtacg ctacaatttc 900
taccctcctt tcctcacccc caaggccatc gcgagctatc acatctttgc cgtcaccgct 960
tccatccctc taagctgcaa agccaaccgc agcggcagcg accttctagc caaagcaaaa 1020
gagagcactt tcttcaaacg cttacctaaa tggcgtctcg gg 1062
Claims (7)
1. an ELISA detection method of differentiating that I group I fowl adenovirus infects, it is characterized in that: taking FAVI100K recombinant protein as envelope antigen, chicken serum is for detecting sample, and the goat-anti chicken IgG of horseradish peroxidase-labeled is that ELIAS secondary antibody detects the antibody that the infection of I group I fowl adenovirus produces;
Described envelope antigen is to prepare through following steps:
(1) prokaryotic expression of FAVI100K recombinant protein: taking the DNA of FAVI CELO strain as template, amplification 100K gene leading portion nucleotide sequence GenBank:U46933:23671-24732nt, be cloned in pGEX-4T-1 prokaryotic expression carrier, in bacillus coli DH 5 alpha, express, the primer of 100K Gene Partial sequence amplification is as follows:
Upstream primer: 5 '-CCG
gAATTCtCATCGAAAATGGCAGACAAGAT-3 ', inserts EcoR I restriction enzyme site;
Downstream primer: 5 '-ATTT
gCGGCCGCcCCGAGACGCCATTTAGGTA-3 ', inserts Not I restriction enzyme site;
(2) qualification to FAVI100K recombinant protein reactionogenicity;
(3) the affinity chromatography method through glutathione agarose resin adsorption, washing, wash-out by the FAVI100K recombinant protein after qualification, the 100K-pGEX recombinant protein of acquisition purifying, i.e. envelope antigen.
2. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 1 infects, is characterized in that: described chicken serum comprises FAVI negative serum, experimental infection serum and inactivated vaccine immune serum.
3. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 1 and 2 infects, is characterized in that comprising the steps:
(1) coated: envelope antigen is diluted to 1～20 μ g/mL with coating buffer, coated 96 hole ELISA Plate, every hole 100 μ L, after 37 DEG C of incubation 1h, are placed in 4 DEG C of 12～16h, and PBST washes plate 3 times, pats dry;
(2) sealing: every hole adds confining liquid 200 μ L, hatches for 37 DEG C, sealing 1h, and PBST washes plate 3 times, pats dry;
(3) be combined with serum: add detection sample, 100 μ L/ holes, establish negative standard items and positive criteria product for contrast, hatch after 1h in 37 DEG C, and PBST washes plate 3 times, pats dry;
(4) be combined with ELIAS secondary antibody: the goat-anti chicken IgG dilution of horseradish peroxidase-labeled is doubly diluted with 1:1000～1:8000, and every hole adds 100 μ L, is placed in 37 DEG C and hatches after 1h, and PBST washes plate 3 times, pats dry;
(5) colour developing: every hole adds 100 μ L substrate nitrite ions, in 37 DEG C of lucifuge effect 10min;
(6) stop: every hole adds 50 μ L stop buffers, cessation reaction;
(7) result is judged: read 450nm wavelength place light absorption value, i.e. OD450 value in microplate reader;
Calculate OD450 mean value X and the standard deviation SD of FAVI negative serum, the X+3SD of FAVI negative serum, as yin and yang attribute critical value, if detect sample OD450 value > yin and yang attribute critical value, is judged to the positive; If sample OD450 value < yin and yang attribute critical value, is judged to feminine gender.
4. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 3 infects, is characterized in that:
(1) described coating buffer is carbonate buffer solution: Na
2cO
31.59g, NaHCO
32.93g, adding distil water is settled to 1000mL, adjust pH to 9.6;
(2) described PBST is the phosphate buffer that contains 0.05% Tween-20: NaCl8.0g, KCl0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o2.9g, Tween-20 500 μ L, adding distil water is settled to 1000mL, adjust pH to 7.4;
(3) described confining liquid is 5% skimmed milk, and 5g skimmed milk is settled to 100mL with PBST;
(4) described dilution is 1%BSA, and 1g bovine serum albumin(BSA) BSA is settled to 100mL with PBST;
(5) described substrate nitrite ion is: TMB damping fluid 10mL, TMB solution 0.5mL, H
2o
232 μ L;
(6) described stop buffer is 2M H
2sO
4, the 21.7mL concentrated sulphuric acid slowly adds in 178.3mL distilled water.
5. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 3 infects, is characterized in that: described negative standard items are for hatching the serum of produced 2～5 Japanese instar chicklings with SPF chicken embryo.
6. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 3 infects, is characterized in that: described positive criteria product comprise inactivated vaccine positive criteria product and experimental infection positive criteria product.
7. the ELISA detection method that discriminating I group I fowl adenovirus according to claim 6 infects, is characterized in that:
Described inactivated vaccine positive criteria product are the SPF chicken serum after twice subcutaneous inoculation of I group I fowl adenovirus inactivated vaccine;
Described experimental infection positive criteria product are the SPF chicken serum after twice of mode collunarium, the eye droppings infecting through I group I fowl adenovirus simulating nature.
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| CN107607717A (en) * | 2017-08-08 | 2018-01-19 | 扬州大学 | The indirect ELISA reagent kit of the detection type aviadenovirus antibody of serum 4 based on F2 albumen |
| CN108752434B (en) * | 2018-07-04 | 2020-07-21 | 扬州大学 | Antigen polypeptide and kit containing same |
| CN112379087B (en) * | 2020-10-21 | 2022-02-11 | 中国医学科学院医学生物学研究所 | Lysate applied to novel coronavirus inactivated vaccine and antigen dissociation method |
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