CN103360472A - Acian Metapneumovirus antibody ELISA detection kit - Google Patents
Acian Metapneumovirus antibody ELISA detection kit Download PDFInfo
- Publication number
- CN103360472A CN103360472A CN2013103251098A CN201310325109A CN103360472A CN 103360472 A CN103360472 A CN 103360472A CN 2013103251098 A CN2013103251098 A CN 2013103251098A CN 201310325109 A CN201310325109 A CN 201310325109A CN 103360472 A CN103360472 A CN 103360472A
- Authority
- CN
- China
- Prior art keywords
- albumen
- protein
- elisa
- subgroup
- metapneumovirus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000351643 Metapneumovirus Species 0.000 title claims abstract description 47
- 238000002965 ELISA Methods 0.000 title claims abstract description 43
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 30
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000011248 coating agent Substances 0.000 claims abstract description 11
- 238000000576 coating method Methods 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 9
- 239000000178 monomer Substances 0.000 claims abstract description 9
- 210000002966 serum Anatomy 0.000 claims description 52
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 238000005406 washing Methods 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 24
- 239000000758 substrate Substances 0.000 claims description 24
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 23
- 239000000872 buffer Substances 0.000 claims description 21
- 238000001962 electrophoresis Methods 0.000 claims description 21
- 239000013612 plasmid Substances 0.000 claims description 21
- 241000287828 Gallus gallus Species 0.000 claims description 19
- 235000018102 proteins Nutrition 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 19
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- 238000007865 diluting Methods 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000003292 glue Substances 0.000 claims description 10
- 235000020183 skimmed milk Nutrition 0.000 claims description 10
- 108010013369 Enteropeptidase Proteins 0.000 claims description 8
- 101150034814 F gene Proteins 0.000 claims description 8
- 230000029087 digestion Effects 0.000 claims description 8
- 239000013642 negative control Substances 0.000 claims description 8
- 239000013641 positive control Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000011156 evaluation Methods 0.000 claims description 7
- 239000013613 expression plasmid Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 238000003259 recombinant expression Methods 0.000 claims description 7
- 238000001262 western blot Methods 0.000 claims description 7
- 102100029727 Enteropeptidase Human genes 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 241001519465 Avian metapneumovirus Species 0.000 claims description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 238000010521 absorption reaction Methods 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- 239000004411 aluminium Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000001742 protein purification Methods 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 3
- 235000011149 sulphuric acid Nutrition 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 20
- 102000036639 antigens Human genes 0.000 abstract description 20
- 108091007433 antigens Proteins 0.000 abstract description 20
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 230000002265 prevention Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000008157 ELISA kit Methods 0.000 abstract 1
- 206010066226 Metapneumovirus infection Diseases 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 239000000047 product Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000004927 fusion Effects 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 206010051497 Rhinotracheitis Diseases 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 230000009089 cytolysis Effects 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 241000711955 Turkey rhinotracheitis virus Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000003149 assay kit Methods 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000272525 Anas platyrhynchos Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000710914 Totivirus Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000002390 adhesive tape Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 108010087302 Viral Structural Proteins Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 208000023406 head swelling Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- -1 washings Substances 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides an Acian Metapneumovirus antibody ELISA detection kit. The kit is the ELISA kit established for detecting the Acian Metapneumovirus antibody on the basis of treating the monomer protein of the A subgroup Acian Metapneumovirus F protein as a coating antigen. The invention also provides a preparation method of the monomer protein of the coating antigen F protein for making the kit. The F protein prepared through the method has a good reactionogenicity, and the established ELISA detection kit provides an effective method for the early-stage diagnosis and detection of the Acian Metapneumovirus infection, and plays a substantial promotion effect in the prevention researches of the Acian Metapneumovirus infected diseases.
Description
Technical field
The present invention relates to the immunological technique field, be specifically related to a kind of monomeric protein of A subgroup fowl metapneumovirus F albumen and for detection of the ELISA test kit of above-mentioned A subgroup fowl metapneumovirus antibody.
Background technology
Fowl metapneumovirus (Avian Metapneumovirus, aMPV), have another name called Turkey Rhinotracheitis Virus (Turkey Rhinotracheitis Virus, TRTV), belonging to Paramyxoviridae Pneumovirinae Pneumovirus, is the bird important pathogen that distributes in a kind of world wide.This virus mainly causes the upper respiratory tract systemic disease of turkey and chicken, such as turkey rhinotracheitis (Turkey Rhinotracheitis, TRT), fowl rhinotracheitis (Avian Rhinotracheitis, ART), and in close relations with the swollen syndrome (Swollen Head Syndrome, SHS) of broiler chicken.Wherein, the turkey rhinotracheitis is a kind of acute height contagious disease of harm turkey aquaculture, this disease take have a running nose, sneeze, under hole swelling under the socket of the eye, head swelling and the laying rate degradation symptom as principal character, infectious rate, sickness rate can be up to 100%, mortality ratio changes with feeding and management and sanitary condition, if polyinfection is arranged, mortality ratio can be up to 25%～40%.This disease transmission is strong, propagates rapidly, can cause multiple bird infection morbidity and cause serious financial loss.At present China is the poultry big country that ranks the first in the world, and 9,600,000,000 of the numbers of animals raised of chicken, the annual mortality ratio of bringing because of all kinds of poultry dieases be up to 20%～25%, loss hundreds of hundred million Renminbi.
At present, serology is the most frequently used method of aMPV diagnosis, particularly vaccination chicken group not.Method commonly used has indirect enzyme-linked immunosorbent adsorption experiment, neutralization of virus, indirect immunofluorescence and immunodiffusion(ID).Wherein, ELISA is the most frequently used method, has quick, sensitive, few, the easy-operating characteristics of amount of serum.Commercialization aMPV ELISA detection kit is from Europe and the U.S. on the market, is mainly used in the detection of serum antibody, and its coated antigen is the totivirus that extracts behind the virus inoculation cell, brings potential harm usually for the propagation of this disease; In addition, these detection reagent are inadequate such as the susceptibility of the antibody test of A subgroup to specific subgroup; Particularly still do not use at present the recombinant protein of expressing to do the development that envelope antigen carries out the ELISA detection kit.AMPV contains multiple virus structural protein, and wherein F albumen is the major structural protein of virus, contains the neutrality epitope that can protect body opposing aMPV to infect, affects recombinant expressed in expression system of this albumen but this F albumen contains a plurality of cross-films district.How expressing the recombinant protein that contains the neutrality epitope sets up the detection that ELISA method exploitation antibody assay kit is used for this disease as envelope antigen and has realistic meaning.At present the plant that infects in the multiple poultry kind of China of fowl metapneumovirus occurs with popular, therefore, develops special, sensitive, easy diagnostic kit and be the active demand that the current prevention and control of China should disease.
Summary of the invention
In order to overcome the defective of prior art, an object of the present invention is for the preparation of the ELISA test kit that detects fowl metapneumovirus antibody, this test kit provides a cover effective instrument for the detection of fowl metapneumovirus antibody.
Another object of the present invention provides a kind of preparation and purification method of monomeric protein of A hypotype fowl metapneumovirus F albumen, for fowl metapneumovirus antibody assay kit provides envelope antigen.
A further object of the present invention is the application of above-mentioned ELISA test kit on rapid detection fowl metapneumovirus.
For realizing above purpose, the present invention at first provides a kind of A hypotype fowl metapneumovirus F albumen, and this albumen is on the basis with A hypotype fowl metapneumovirus F albumen wild-type, removes its part cross-film district and obtains, and its aminoacid sequence is shown in SEQ ID No.2.The present invention also comprises the gene of encoding sequence, and its nucleotide sequence is shown in SEQ ID No.1.The research discovery, this albumen has good immune response originality, can be used for the antibody test of aMPV.
Accordingly, the invention provides a kind of fowl metapneumovirus antibody ELISA detection kit, it comprises the coated elisa plate of monomeric protein of A type fowl metapneumovirus F albumen.
The ELISA test kit of above-mentioned detection fowl metapneumovirus also comprises ELIAS secondary antibody, positive control, negative control, washings, sample diluting liquid, substrate solution A, substrate solution B and stop buffer.
Wherein, described ELIAS secondary antibody is horseradish peroxidase mark goat-anti chicken monoclonal antibody; Described positive control is the chicken serum of natural infection A subgroup avian pneumovirus; Described negative control is normal chicken serum; Described coating buffer, washings, confining liquid, sample diluting liquid, substrate solution A, component and the proportioning of substrate solution B and stop buffer are as follows:
Coating buffer: anhydrous Na
2CO
31.59g, NaHCO
32.93g adding distil water is to 1000mL;
Washings: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g, Tween-200.5mL adds distilled water to 1000mL;
Confining liquid: 5% skim-milk;
Sample diluting liquid: the washings that contains 5% skim-milk;
Substrate solution A: urea peroxide 1.0g, Na
2HPO
412H
2O35.8g, citric acid H
2O10.3g, Tween-20100 μ L adds 1000mL distilled water;
Substrate solution B:TMB700mg, DMSO40mL, fully dissolving, adding distil water 960mL then, citric acid H
2O10.3g;
Stop buffer: 2mol/L sulphuric acid soln.
Above-mentioned for the monomeric protein preparation of ELISA test kit as the A type fowl metapneumovirus F albumen of enzyme plate envelope antigen, may further comprise the steps: synthetic A subgroup fowl metapneumovirus F protein gene sequence is that template amplification obtains the sequence shown in the SEQ ID No.1, the above-mentioned purpose gene is imported in the pBCX carrier, product after the connection transforms DH5 α competent cell, evaluation and screening goes out positive recombinant expression plasmid pBCX-aMPV-A-F after extracting the plasmid double digestion, with abduction delivering behind the above-mentioned positive recombinant expression plasmid conversion BL21 competent cell, SDS-PAGE electrophoretic analysis expressing protein, adopt again Ni post affinity chromatography method, target protein is carried out purifying, then detect the protein purification result with SDS-PAGE and Western blot respectively, target protein behind the purifying is processed with enteropeptidase, cut the result with SDS-PAGE check enzyme after processing, cutting glue with the KCl staining after electrophoresis finishes reclaims, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify, elisa assay is cut the antigenicity that glue reclaims albumen.
Particularly, the preparation of the monomeric protein of A type fowl metapneumovirus F albumen can may further comprise the steps:
1) amplifying target genes.Take synthetic A subgroup fowl metapneumovirus F protein gene sequence as template, by PCR method its F gene is increased, clones and obtain goal gene (F gene, its disappearance part cross-film district), upstream primer adds Kpn I restriction enzyme site, downstream primer adds Hind III restriction enzyme site, and primer sees Table 1:
The amplimer of table 1F gene
2) carry out double digestion with Kpn I, Hind III behind the recovery of F gene and the PBCX plasmid extraction, reclaim the purpose fragment, under 16 ° of C, connect 12-16h under the effect of T4DNA ligase enzyme goal gene being connected 5:1 with carrier, transformed competence colibacillus cell DH5 α, extract plasmid, after Kpn I and the evaluation correctly of Hind III double digestion, obtain positive recombinant expression plasmid pBCX-aMPV-A-F.
3) then get plasmid pBCX-aMPV-A-F and transform the BL21 competent cell, the positive plasmid bacterium that obtains is cultivated in 37 ° of C, treat that the A600 value reaches at 0.4～0.6 o'clock, adding IPTG is that 1.2mmol/L carries out abduction delivering to final concentration, the thalline of 4h behind the collection abduction delivering, ultrasonic disruption is got precipitation after centrifugal and is carried out the SDS-PAGE electrophoretic analysis.
4) then adopt Ni post affinity chromatography method, the purifying target protein, target protein behind the purifying carries out SDS-PAGE and Western blot identifies, process with enteropeptidase through the F albumen of identifying, then carry out the SDS-PAGE electrophoresis, take off gel after electrophoresis finishes, first with the distilled water washing, then immerse the 5min that develops the color in the KCl solution of 250mmol/L of 4 ° of C precoolings, the target protein band that is painted silvery white is downcut, PBS washing 3 times moves into adhesive tape in the one clean pouch and to pulverize, and broken end is scraped in the EP pipe, add PBS concussion mixing multigelation 3 times, the centrifugal 2min of 12000r/min draws supernatant, and the SDS-PAGE electrophoresis carries out recovering effect to be identified.
5) elisa assay is cut the antigenicity that glue reclaims albumen.
The present invention also provides the preparation method of mentioned reagent box, comprise the preparation of the elisa plate of A subgroup fowl metapneumovirus F albumen, its method is: with carbonate (pH9.6, concentration 0.5mol/L) damping fluid is made coating buffer, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use again.
Based on test kit of the present invention, can adopt following ELISA method, detect A subgroup fowl metapneumovirus antibody:
1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 30min;
2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 30min;
4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15～20min;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: the absorption photometric value (OD450 value) of measuring every hole with microplate reader at the 450nm place.
The invention provides and use solubility pBCX carrier, expressed the F albumen in A subgroup fowl metapneumovirus disappearance part cross-film district, and utilize 6 * His label of pBCX carrier, the fusion rotein of expressing is carried out purifying, the albumen that Western-blot detects the proof purifying has reactionogenicity, cut with the albumen of enteropeptidase to purifying afterwards, obtain to remove the monomeric protein of carrier label, obtained the higher albumen of immunogenicity.This protein constructs provided by the invention is to have solved the problem of F protein monomer purification difficult.
The present invention is prepared from as the basis take the F albumen of gene engineering expression, and the F protein monomer is non-totivirus antigen, and security is good, does not contain irrelevant foreign protein, and has good antigenicity, and the detection kit of therefore inventing has very high specificity and susceptibility.
The present invention makes up is used for the test kit that A subgroup fowl metapneumovirus detects, easy to operation, detects responsively fast, is specially adapted to clinically the rapid detection to fowl metapneumovirus antibody, for the Prevention Research of this disease has played remarkable promoter action.
Description of drawings
Fig. 1 is the electrophorogram of F gene amplification, the purpose band of 1:F albumen wherein, 2: blank, M:MarkerDL2000.
Fig. 2 is the recombinant plasmid PCR evaluation figure that the F gene clone obtains to the pBCX carrier, M:DNA MarkerDL2000 wherein, 1～10: the recombinant plasmid pcr amplified fragment.
The enzyme of Fig. 3 recombinant plasmid pBCX-F that to be the F gene clone obtain to the pBCX carrier is cut evaluation figure, M:DNAMarkerDL2000 wherein, 1～3: the endonuclease bamhi of positive recombinant plasmid pBCX-F.
Fig. 4 is after pBCX-aMPV-A-F is transformed into the BL21 competent cell, extracts plasmid PCR and identifies figure, M:DNAMarkerDL2000 wherein, 1: the PCR product of recombinant plasmid, 3: blank.
Fig. 5 is the SDS-PAGE analysis chart of induction expression protein, M:175Marker wherein, and 1: the empty plasmid expression product, 2～8 are respectively the bacteria lysis product that 1.2mmol/LIPTG induces 0～6h.
Fig. 6 is the soluble analysis evaluation figure of fusion rotein, M:175Marker wherein, 1: bacteria lysis gross product, 2: the bacteria lysis precipitation; 3: the bacteria lysis supernatant.
Fig. 7 is the SDS-PAGE analysis chart of purifying protein, M:175Marker wherein, 1～4: the pipe of the 1st to 4 behind purifying wash-out product.
Fig. 8 is the Western-blot analysis chart of purifying protein, (a) middle M:175Marker, 1: be the fusion protein product of purifying; (b) M:175Marker in, 1,2,3: for being the fusion protein product of purifying.
Fig. 9 is that enzyme is cut rear KCl dyeing and cut the qualification result that glue reclaims target protein, M:175Marker wherein, 1,2: be respectively two pipe products after enzyme cuts back to close.
Embodiment
Following examples are used for further specifying of the present invention, but should not be construed limitation of the present invention.Under the prerequisite that does not deviate from the present invention's spirit and essence, modification or replacement to the present invention does all belong to category of the present invention.
Preparation and the purifying of the protein monomer of embodiment 1A subgroup fowl metapneumovirus F albumen
The gene order of the A subgroup fowl metapneumovirus F albumen shown in the synthetic SEQ ID NO.1, with a pair of Auele Specific Primer of Oligo6.0 software design, upstream primer adds Kpn I restriction enzyme site, and downstream primer adds Hind III restriction enzyme site, and upstream primer is: 5 '-GG
GGTACCGAGGCAGTATCCACATTAGGG-3 ', downstream primer is: 5 '-CCC
AAGCTTCTTGGCATCTGCACCTAG-3 '.(ddH increases in 20 μ L reaction systems take pcmy-myc-L plasmid (being purchased from Invitrogen company) as template
2O11.5 μ L, 5 * Buffer4 μ L, Taq enzyme 0.2 μ L, dNTP2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, dna profiling 0.3 μ L). amplification condition is: 94 ° of C denaturation 3min; 94 ° of C sex change 30s, 59 ° of C annealing 45s, 72 ° of C extend 1.5min, totally 35 circulations; 72 ° of C extend 10min; 4 ° of C preserve (amplification is seen Fig. 1).
Get 100 μ L PCR products and carry out agarose gel electrophoresis, 100V electrophoresis 35～40min, ultraviolet detection product analysis result, and cut glue and reclaim.The purpose fragment through the Kpn I be connected with Hind III double digestion with the plasmid of being connected with Hind III double digestion through the Kpn I in molar ratio 5:1 under 16 ° of C, be connected 12～16h, transform DH5 α, 37 ° of C overnight incubation.Choosing colony extracts plasmid, uses Auele Specific Primer to carry out pcr amplification, and with above-mentioned restriction endonuclease digestion, electrophoresis is identified (the results are shown in Figure 2).Positive bacterium colony after identifying correctly checks order through plasmid and further verifies, and confirms that its gene order is correct, and the correct recombinant expression plasmid called after pBCX-aMPV-A-F(of order-checking be the results are shown in Figure 3).
From the correct bacterium colony that checks order, extract Plasmid Transformation BL21 competent cell, obtain recombinant bacterium (the results are shown in Figure 4), in the LB liquid nutrient medium, cultivate recombinant bacterium, treat that bacterium liquid is at 600nm optical density(OD) (optical density, OD) value under reaches at 0.4～0.6 o'clock, it is 1.2mmol/L that adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) makes its final concentration, abduction delivering.Collect the thalline that different induction times are expressed, ultrasonic disruption, centrifugally get respectively cleer and peaceful precipitation and carry out the SDS-PAGE electrophoresis afterwards, the result shows and induces the target protein overwhelming majority that reaches maximum (the results are shown in Figure 5) behind the 4h and merge to express (the results are shown in Figure 6) with soluble form.
After fusion rotein is defined as secretion type expression, directly the fusion rotein bacterial lysate is carried out purifying with the Ni-NTA purification column to target protein, with elutriant wash-out purification column, collect elutriant, contain the purpose fusion rotein in the elutriant, the F albumen that removes part cross-film district adds the label protein on the carrier, and the albumen size after the fusion is 74kDa, measure the concentration of albumen in the elutriant with ultraviolet spectrophotometer, the highest one is 27mg/mL (the results are shown in Figure 7).
Through the SDS-PAGE electrophoresis, utilize Western-blot that the fusion rotein of purifying is carried out the reactionogenicity analysis, primary antibodie is the monoclonal antibody of A hypotype aMPV F albumen, two anti-be the goat anti-mouse IgG of HRP mark, detected result shows: a darker purpose band is arranged at the 74kDa place, the fusion rotein of this explanation restructuring has reactionogenicity, and the albumen that the size of purpose band and SDS-PAGE express size position consistency, band is single, the WB detected result of albumen before the WB detected result of albumen behind the purifying and the purifying is compared, can prove fusion protein purification success (the results are shown in Figure 8).
F albumen is processed with enteropeptidase, then carry out the SDS-PAGE electrophoresis, electrophoresis takes off gel after finishing, wash with distilled water first, then immerse the 5min that develops the color in the KCL solution of 250mmol/L of 4 ° of C precoolings, the target protein band that is painted silvery white is downcut, PBS washing 3 times, adhesive tape moved in the clean pouch pulverize, broken end is scraped in the EP pipe, add PBS concussion mixing multigelation 3 times, the centrifugal 2min of 12000r/min, draw supernatant, the SDS-PAGE electrophoresis carries out recovering effect and identifies (the results are shown in Figure 9).
The Immunity identification of the F protein monomer that embodiment 2 the present invention make up
The F albumen that enzyme is cut back to close and the F albumen cut without enzyme are diluted to coated 96 orifice plates of 10 μ g/mL with the 0.5mol/L carbonate buffer solution of pH9.6, take fowl metapneumovirus infected chicken positive serum and negative serum as primary antibodie takes turns doing 3 gradient dilutions, be respectively 1:100,1:300,1:500.Take the anti-chicken IgG of the goat of HRP mark as two anti-, working concentration is 1:10000; Each hole OD after the TMB colour developing, is read in parallel 2 holes of doing of each sample
450nmValue, result show: without the enteropeptidase enzyme cut and with the albumen of carrier label as the elisa plate envelope antigen, when detecting serum, the OD value of negative serum is between 0.6-0.8, near the OD value of positive serum; And process enteropeptidase enzyme only keeps monomeric protein as the envelope antigen of elisa plate after cutting processing, when detecting serum, negative OD value is between 0.1-0.2, the P/N value of positive OD value and negative OD value is between 3.6-4.45, as seen ELISA result is good, the monomeric protein that enzyme cuts back to close has good reactionogenicity, and detected result sees Table 2:
Table 2ELISA detects the antigenicity that the KCl staining is cut glue purification albumen
The foundation of embodiment 3 fowl metapneumovirus ELISA detection methods
1.A determining of subgroup fowl metapneumovirus antibody ELISA detection kit optimum reaction condition
1) the best coated concentration of antigen and enzyme mark monoclonal antibody best effort concentration determines
Take the F monomeric protein of purifying as envelope antigen, the goat-anti chicken monoclonal antibody of HRP mark is second antibody, carries out the square formation burette test at enzyme plate.Make respectively 15 μ g/ml, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL with the F albumen that carbonate buffer solution cuts back to close enzyme, dilute 4 extent of dilution, each extent of dilution repeats 2 holes, 100 μ L/ holes, and 4 ℃ of coated elisa plates spend the night.Feminine gender and the positive serum of chicken are made respectively 1:100,1:300,1:500 doubling dilution, and each extent of dilution repeats 2 holes, and 100 μ L/ holes form the coated concentration of the best and primary antibodie thinning ratio row that square formation is determined recombinant protein.Behind the sealing 1h, each extent of dilution repeats 2 holes, 100 μ L/ holes, and each goes on foot 37 ℃ of reaction 30min, stops behind 37 ℃ of colour developing 5min, measures each hole OD in microplate reader
450nmValue.As can be seen from Table 3, antigen coated amount is the 1000ng/ hole, and the serum weaker concn is considered as best results when being 1:100, (as calculated, when the coated concentration of albumen be the 1000ng/ hole, when serum dilution is 1:100, OD
450nmRecord absorbancy P/N value maximum)
Determining of the antigen coated concentration of table 3 and serum dilution
2) selection of best coated condition
With the antigen coated concentration coated elisa plate of the best, each coated concentration repeats 2 holes, respectively with 4 ° of C spend the night, behind 37 ° of C effect 1h again 4 ° of C spend the night coatedly, measure each hole OD
450nmValue is calculated the P/N value and is selected best coated condition.As can be seen from Table 4, behind 37 ° of C1h 4 ° of C to spend the night be the coated condition of the best.
The selection of the coated condition of table 4
3) selection of confining liquid and off-period
With the suitableeest antigen coated concentration coated elisa plate, with the coated condition envelope antigen of the best, test with following three kinds of confining liquids respectively: 5% skimmed milk, 5% calf serum, 1%BSA, each confining liquid repeats 2 holes; With following 3 conditions sealing, test: 37 ° of C30min, 37 ° of C1h, 37 ° of C1.5h, 37 ° of C2h, each sealing condition repeats 2 holes respectively.Survey each hole OD
450Value is calculated the P/N value.As can be seen from Table 5, the 2h5% skimmed milk is top condition.
Determining of table 5 sealing condition
4) two anti-optimum dilution degrees and the selection in reaction times
With the coated condition of the best, by the suitableeest antigen coated concentration coated elisa plate, use best confining liquid under best sealing condition, to seal enzyme plate, two anti-ly press respectively 1:10000,1:20000,1:30000,1:40000,1:50000 dilution, 37 ° of C are respectively 30min, 1h, 1.5h and 2h lower action time, each two anti-condition repeats 2 holes, records each hole OD
450nmValue is calculated the P/N value.As can be seen from Table 6,1:4000030min is best two anti-activity and the times.
Table 6 two anti-concentration and the selection of two anti-action times
5) selection of substrate developing time
With the coated condition of the best, by the suitableeest antigen coated concentration coated elisa plate, under best confining liquid and sealing condition, respectively at color development at room temperature 10min, 15min, 20min, 25min.Record each hole OD
450nmValue is calculated the P/N value, can find out according to table 7, and the P/N value was larger when developing time was 15min, and positive the value about 1.0, and negative value is top condition about 0.1, be best substrate developing time therefore select colour developing 15min.
Determining of table 7 substrate developing time
6) the Cut-off value determines
Select 20 parts of negative chicken serums, under the suitableeest antigen coated concentration and serum dilution, carry out the ELISA reaction under above definite optimum reaction condition, the result is as shown in table 8:
The measurement result of table 820 part negative chicken serum
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD 450 | 0.157 | 0.172 | 0.177 | 0.18 | 0.202 | 0.207 | 0.179 | 0.157 | 0.172 | 0.18 |
| Serum | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| OD 450 | 0.177 | 0.173 | 0.158 | 0.182 | 0.108 | 0.118 | 0.179 | 0.142 | 0.121 | 0.137 |
The mean value X=0.1639 of 20 parts of serum, standard deviation SD=0.026052, according to formula, positive cut-off value is 0.2419(Cp=X+3SD), the cut-off value of feminine gender is 0.2159(Cn=X+2SD).With formula S/P=(S-N)/(P-N) come result of determination, if S/P 〉=Cp, serum to be checked is positive; If S/P＜Cn, serum to be checked is negative; If Cp＞S/P 〉=Cn is judged to suspiciously, need redeterminate.(S represents the OD450 value of sample to be checked; P represents the OD of positive control
450Value; N represents the OD of negative control
450Value).
7) repeated experiment between plate
Get the coated enzyme plate of 4 different batches, detect 4 parts of known positive serums, every block of plate of each sample arranges 2 repetitions, calculates interassay coefficient of variation CV=(SD/OD450nm mean value) * 100%, the results are shown in Table 9:
Determining of table 9 interassay coefficient of variation
OD450nm mean value=0.963969, SD=0.07474 calculates interassay coefficient of variation CV=(SD/OD450nm mean value according to formula) * 100%=7.75%, CV＜10%, illustrate that the degree of variation of same serum sample between different plates is very little, have good stability.
8) repeated experiment in the plate
Repeat in the same enzyme plate is criticized, detect 4 parts of known positive serums, each sample arranges 4 repetitions, calculates variation within batch coefficient CV=(SD/OD450nm mean value) * 100%, the results are shown in Table 10:
Determining of table 10 variation within batch coefficient
| The chicken serum numbering | Positive value (P) | The chicken serum numbering | Positive value (P) | N |
| A1 | 0.947 | B1 | 0.947 | 0.13 |
| A2 | 0.958 | B2 | 0.998 | 0.167 |
| A3 | 0.944 | B3 | 0.924 | 0.145 |
| A4 | 0.950 | B4 | 1.021 | 0.129 |
| C1 | 1.097 | D1 | 0.896 | 0.121 |
| C2 | 1.102 | D2 | 0.898 | 0.161 |
| C3 | 1.052 | D3 | 0.894 | 0.17 |
| C4 | 1.087 | D4 | 0.890 | 0.168 |
OD450nm mean value=0.975313, SD=0.072513 calculates interassay coefficient of variation CV=(SD/OD450nm mean value according to formula) * 100%=7.43%, CV＜10%, illustrate that the degree of variation of different serum samples in same plate is very little, have good repeatability.
9) specificity of indirect ELISA method experiment
Utilize the indirect ELISA method of setting up, detect positive serum, control group adopts AEV/ND/REO/IBD/IB serum and negative serum, positive serum OD
450nmNear 1.0, negative serum OD
450nmAt 0.1-0.2, control group serum OD
450nmAt 0.1-0.2, prove that the indirect ELISA method specificity of setting up with this albumen is better, the results are shown in Table 11:
The specificity of table 11 indirect ELISA method
10) sensitivity experiments of indirect ELISA method
One antiserum(antisera) is diluted in the ratio of 1:200,1:400,1:600,1:800,1:1000, to determine its susceptibility.Still can detect the positive when data results is presented at Dilution ratio and reaches 1:1000, prove that the susceptibility of this method is fine, the results are shown in Table 12:
The susceptibility of table 12 indirect ELISA method
| Dilution ratio | Positive serum | Negative serum |
| 1:200 | 1.45 | 0.203 |
| 1:400 | 1.278 | 0.167 |
| 1:600 | 0.969 | 0.142 |
| 1:800 | 0.464 | 0.107 |
| 1:1000 | 0.33 | 0.096 |
11) comparative experiments of indirect ELISA method
With ELISA method and the American I DEXX aMPV of the company antibody assay kit (ELISA) set up 164 parts of clinical censorship serum are carried out Parallel testing, the result shows, the positive coincidence rate of the two is 96.77% (120/124), negative match-rate is 92.5% (37/40), total coincidence rate reaches 95.73% (157/164), the results are shown in Table 13 to table 16, the result shows that the indirect ELISA method of foundation has higher specificity and susceptibility.
The ELISA detected result of 40 parts of serum of table 13 Nanning sampling
The ELISA detected result of the 46 parts of chicken serums in Daxing, table 14 Beijing
The ELISA detected result of 40 parts of serum of table 15 Pinggu County, beijing
The ELISA detected result of the 38 parts of serum in Haidian, table 16 Beijing
The assembling of embodiment 4 fowl metapneumovirus antibody ELISA detection kits
1.ELISA test kit assembling
⑴ 96 hole enzyme plates: the monomeric protein that is coated with F albumen;
⑵ standard positive control: infect fowl metapneumovirus duck positive serum;
⑶ standard negative control: a normal health duck serum;
⑷ the goat-anti chicken monoclonal antibody of horseradish peroxidase-labeled;
⑸ coating buffer: anhydrous Na
2CO
31.59g, NaHCO
32.93g adding distil water is to 1000ml;
⑹ washings: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g, Tween200.5mL adds distilled water to 1000mL;
(7) substrate solution A: urea peroxide 1.0g, Na
2HPO
412H
2O35.8g, citric acid H
2O10.3g, Tween-20100 μ L adds 1000mL distilled water;
(8) substrate solution B:TMB700mg, DMSO40mL, fully dissolving, adding distil water 960mL then, citric acid H
2O10.3g;
(9) confining liquid: 5% skim-milk;
(10) diluent: the washings that contains 5% skim-milk; ⑼ stop buffer: 2mol/L sulphuric acid soln.
2. the preparation of enzyme plate
With carbonate (pH9.6, concentration 0.5mol/L) damping fluid is made coating buffer, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use again.
The mensuration program of embodiment 5A hypotype fowl metapneumovirus antibody ELISA detection kit
1. reagent is prepared and diluted sample
1) reagent need not preparation in the test kit of the present invention, can directly use;
2) dilution of sample to be checked and standard reference material: the standard positive, the standard negative reference substance that provide in serum sample to be detected and the test kit are used afterwards with 10 times of sample diluting liquid dilutions.
2. determination step
1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 60min;
2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 60min;
4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15min;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: use microplate reader at OD
450nmThe place measures the absorption photometric value (OD in every hole
450nmValue).
3. the result judges
Calculate mean number (X) and the standard deviation (SD) of its absorbance, then positive Cut-off value Cp=X+3SD; Negative Cut-off value Cn=X+2SD.Using formula S/P=(S – N)/(P – N) comes result of determination (S: the OD of sample to be checked
450nmValue; P: the OD of positive control
450nmValue; N: the OD of negative control
450nmValue).If S/P 〉=Cp, serum to be checked is positive; If S/P＜Cn, serum to be checked is negative; If Cp＞S/P 〉=Cn is judged to suspiciously, need redeterminate.(S represents the OD of sample to be checked
450nmValue; P represents the OD of positive control
450nmValue; N represents the OD of negative control
450nmValue).
Claims (12)
1.A subgroup fowl metapneumovirus F albumen, its aminoacid sequence is shown in SEQ ID No.2.
2. the gene of coding claim 1 described F albumen.
3. gene according to claim 2, its nucleotide sequence is shown in SEQ ID No.1.
4. the ELISA detection reagent that contains the described A subgroup of claim 1 fowl metapneumovirus F albumen.
5. A subgroup fowl metapneumovirus antibody ELISA detection kit, it comprises the elisa plate of coated A subgroup fowl metapneumovirus F albumen claimed in claim 1.
6. test kit according to claim 5 is characterized in that, also comprises in ELIAS secondary antibody, sample diluting liquid, concentrated cleaning solution, substrate solution A, substrate solution B, stop buffer, positive control and the negative control one or more.
7. ELISA detection kit according to claim 6 is characterized in that, described ELIAS secondary antibody is horseradish peroxidase-labeled goat-anti chicken monoclonal antibody; Described positive control is the chicken serum of natural infection A subgroup avian pneumovirus; Described negative control is normal chicken serum; Contain NaCl8.0g, KH in every liter of the described washings
2PO
40.2g, Na2HPO
412H
2O2.9g, KCl0.2g, Tween-200.5mL; Described sample diluting liquid is the washings that contains 5% skim-milk; Contain urea peroxide 1.0g, Na among every liter of the described substrate solution A
2HPO
412H
2O35.8g, citric acid H
2O10.3g, Tween-20100 μ L; Contain TMB700mg, DMSO40mL, citric acid H among every liter of the substrate solution B
2O10.3g; Described stop buffer is the 2mol/L sulphuric acid soln.
8. prepare the method for the described A subgroup of claim 1 fowl metapneumovirus F albumen, it may further comprise the steps:
Synthetic A subgroup fowl metapneumovirus F protein gene sequence is that template amplification obtains the sequence shown in the SEQ ID No.1, the above-mentioned purpose gene is imported in the pBCX carrier, product after the connection transforms DH5 α competent cell, evaluation and screening goes out positive recombinant expression plasmid pBCX-aMPV-A-F after extracting the plasmid double digestion, with abduction delivering behind the above-mentioned positive recombinant expression plasmid conversion BL21 competent cell, SDS-PAGE electrophoretic analysis expressing protein, adopt again Ni post affinity chromatography method, target protein is carried out purifying, then detect the protein purification result with SDS-PAGE and Western blot respectively, target protein behind the purifying is processed with enteropeptidase, cut the result with SDS-PAGE check enzyme after processing, cutting glue with the KCl staining after electrophoresis finishes reclaims, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify, elisa assay is cut the antigenicity that glue reclaims albumen.
9. the preparation method of A subgroup fowl metapneumovirus F albumen according to claim 8, it may further comprise the steps:
1) take synthetic A subgroup fowl metapneumovirus F protein gene sequence as template, by PCR method its F gene is increased, clones and obtain goal gene;
The upstream and downstream primer of amplification F gene is respectively:
Upstream primer: 5 '-GG
GGTACCGAGGCAGTATCCACATTAGGG-3 '
Downstream primer: 5 '-CCC
AAGCTTCTTGGCATCTGCACCTAG-3 ';
2) simultaneously goal gene and pBCX carrier being carried out enzyme with Kpn I, Hind III cuts, reclaim the purpose fragment, 16 ° of C connections are spent the night, and transform DH5 α competent cell, extract plasmid, after Kpn I and the evaluation correctly of Hind III double digestion, obtain positive recombinant expression plasmid pBCX-aMPV-A-F;
3) then get plasmid pBCX-aMPV-A-F and transform the BL21 competent cell, the positive plasmid bacterium that obtains is cultivated in 37 ° of C, treat that the A600 value reaches at 0.4～0.6 o'clock, adding IPTG is that 1.2mmol/L carries out abduction delivering to final concentration, the thalline of 4h behind the collection abduction delivering, ultrasonic disruption is got precipitation after centrifugal and is carried out the SDS-PAGE electrophoretic analysis;
4) then adopt Ni post affinity chromatography method, the purifying target protein, target protein behind the purifying carries out SDS-PAGE and Westernblot identifies, process with enteropeptidase through the F albumen of identifying, then carry out the SDS-PAGE electrophoresis, cut glue with the KCl staining after electrophoresis finishes and reclaim, the F albumen that reclaims gained is carried out recovering effect through the SDS-PAGE electrophoresis identify;
5) elisa assay is cut the antigenicity that glue reclaims albumen.
10. the preparation method of each described test kit of claim 5～7, it comprises the elisa plate that adopts the coated A subgroup fowl metapneumovirus F albumen claimed in claim 1 of following steps preparation: make coating buffer with carbonate buffer solution, with above-mentioned coating buffer the F protein monomer being diluted is 10 μ g/mL, add in the ELISA Sptting plate by 100 μ L/ holes, 37 ° of C sealed 1 hour, 4 ° of coated spending the night of C, pat dry, again with 37 ° of C sealings of 5% skimmed milk 2 hours, to contain the PBST washing of 0.05% tween, pat dry with aluminium film vacuum sealing preservation, for subsequent use.
11. the application of the described ELISA detection kit of claim 5～7 any one on rapid detection A subgroup fowl metapneumovirus antibody.
12. application according to claim 11 is characterized in that, may further comprise the steps:
1) application of sample: add the serum sample to be detected 100 μ L after doubly diluting with sample diluting liquid 1:100 in the enzyme plate micropore, 37 ° of C constant-temperature incubation 30min;
2) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
3) add enzyme labelled antibody: add enzyme labelled antibody working fluid 100 μ L in every hole, 37 ° of C constant-temperature incubation 30min;
4) washing: pour out the liquid in the hole, add washings 250 μ L in every hole, wash 3 times and pat dry;
5) add substrate solution: every Kong Zhongxian adds substrate solution A50 μ L, adds substrate solution B50 μ L again, lucifuge colour developing 15～20min;
6) add stop buffer: add stop buffer 50 μ L in every hole;
7) measure: the absorption photometric value of measuring every hole with microplate reader at the 450nm place.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310325109.8A CN103360472B (en) | 2013-07-30 | 2013-07-30 | Acian Metapneumovirus antibody ELISA detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310325109.8A CN103360472B (en) | 2013-07-30 | 2013-07-30 | Acian Metapneumovirus antibody ELISA detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103360472A true CN103360472A (en) | 2013-10-23 |
| CN103360472B CN103360472B (en) | 2015-02-25 |
Family
ID=49362803
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310325109.8A Expired - Fee Related CN103360472B (en) | 2013-07-30 | 2013-07-30 | Acian Metapneumovirus antibody ELISA detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103360472B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911153A (en) * | 2015-07-10 | 2015-09-16 | 青岛易邦生物工程有限公司 | Method for separating acian metapneumovirus |
| CN105177183A (en) * | 2015-09-18 | 2015-12-23 | 中国农业科学院哈尔滨兽医研究所 | Method for detecting RT-PCR of avian metapneumovirus |
| CN105296439A (en) * | 2015-09-08 | 2016-02-03 | 北京市农林科学院 | Chicken C-type acian metapneumovirus strain(aMPV-JCX) and application thereof |
| CN105693829A (en) * | 2016-04-14 | 2016-06-22 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
| CN106546732A (en) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method |
| CN107904326A (en) * | 2017-12-12 | 2018-04-13 | 北京市农林科学院 | Nucleic acid and application for RT PCR fast typings detection fowl metapneumovirus hypotype |
| CN109001450A (en) * | 2018-04-24 | 2018-12-14 | 中国农业科学院兰州兽医研究所 | A kind of kit and preparation method detecting chicken Mycoplasma synoviae antibody |
| CN105296440B (en) * | 2015-09-08 | 2019-01-04 | 北京市农林科学院 | One breeder c-type fowl metapneumovirus strain aMPV/C-JCZ and its application |
| CN110596400A (en) * | 2019-09-02 | 2019-12-20 | 佛山市正典生物技术有限公司 | Indirect ELISA detection method based on recombinant R7 protein of leucocytozoon casseliflavus and application |
| CN112881682A (en) * | 2021-01-11 | 2021-06-01 | 广西大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for avian metapneumovirus antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921732A (en) * | 2001-01-19 | 2010-12-22 | 维洛诺瓦蒂夫公司 | A virus causing respiratory tract illness in susceptible mammals |
-
2013
- 2013-07-30 CN CN201310325109.8A patent/CN103360472B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921732A (en) * | 2001-01-19 | 2010-12-22 | 维洛诺瓦蒂夫公司 | A virus causing respiratory tract illness in susceptible mammals |
Non-Patent Citations (3)
| Title |
|---|
| I. TARPEY ET AL.: "Cloning, expression and immunogenicity of the avian pneumovirus (Colorado isolate) F protein.", 《AVIAN PATHOLOGY》 * |
| MARIO H. SKIADOPOULOS ET AL.: "Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity.", 《VIROLOGY》 * |
| RENE ALVAREZ, M. ET AL.: "Development of a Nucleoprotein-Based Using a Synthetic Peptide Antigen for Antibodies in Turkey Sera.", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911153A (en) * | 2015-07-10 | 2015-09-16 | 青岛易邦生物工程有限公司 | Method for separating acian metapneumovirus |
| CN105296439B (en) * | 2015-09-08 | 2019-01-04 | 北京市农林科学院 | One breeder c-type fowl metapneumovirus strain aMPV/C-JCX and its application |
| CN105296439A (en) * | 2015-09-08 | 2016-02-03 | 北京市农林科学院 | Chicken C-type acian metapneumovirus strain(aMPV-JCX) and application thereof |
| CN105296440B (en) * | 2015-09-08 | 2019-01-04 | 北京市农林科学院 | One breeder c-type fowl metapneumovirus strain aMPV/C-JCZ and its application |
| CN106546732A (en) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method |
| CN105177183B (en) * | 2015-09-18 | 2018-10-16 | 中国农业科学院哈尔滨兽医研究所 | The method for detecting the RT-PCR of fowl metapneumovirus |
| CN105177183A (en) * | 2015-09-18 | 2015-12-23 | 中国农业科学院哈尔滨兽医研究所 | Method for detecting RT-PCR of avian metapneumovirus |
| CN105693829A (en) * | 2016-04-14 | 2016-06-22 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
| CN105693829B (en) * | 2016-04-14 | 2022-03-15 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
| CN107904326A (en) * | 2017-12-12 | 2018-04-13 | 北京市农林科学院 | Nucleic acid and application for RT PCR fast typings detection fowl metapneumovirus hypotype |
| CN107904326B (en) * | 2017-12-12 | 2021-06-22 | 北京市农林科学院 | Nucleic acid for RT-PCR rapid typing detection of avian metapneumovirus subtype and application |
| CN109001450A (en) * | 2018-04-24 | 2018-12-14 | 中国农业科学院兰州兽医研究所 | A kind of kit and preparation method detecting chicken Mycoplasma synoviae antibody |
| CN110596400A (en) * | 2019-09-02 | 2019-12-20 | 佛山市正典生物技术有限公司 | Indirect ELISA detection method based on recombinant R7 protein of leucocytozoon casseliflavus and application |
| CN112881682A (en) * | 2021-01-11 | 2021-06-01 | 广西大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for avian metapneumovirus antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103360472B (en) | 2015-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103360472B (en) | Acian Metapneumovirus antibody ELISA detection kit | |
| CN102636644B (en) | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen | |
| CN105296441B (en) | A kind of bovine viral diarrhea virus strain and its application | |
| CN103675274A (en) | Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody | |
| CN103543261B (en) | Cattle and sheep Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit and preparation method thereof | |
| CN102967710A (en) | Competitive ELISA kit for peste-des-petits-ruminants antibody detection and preparation method thereof | |
| CN104316703B (en) | A kind of Mycoplasma bovis test strip and its preparation method | |
| CN102175867A (en) | Enzyme-linked immunoassay kit for detecting rabies virus antibody | |
| CN101533016A (en) | Indirect ELISA reagent kit for detecting antibodies against rabbit hemorrhagic disease virus | |
| CN106405093A (en) | Detection kit for detecting infectious bovine rhinotracheitis virus antibody | |
| CN102093999A (en) | Indirect ELISA kit for detecting avian infectious bronchitis virus antibody | |
| CN102023217A (en) | Double-antibody biotin-Avidin ELISA (enzyme-linked immuno sorbent assay) detection kit for cattle viral diarrhea virus and application method thereof | |
| CN104262484A (en) | Specific IgY antibody for resisting African swine fever virus as well as preparation method and application thereof | |
| CN106405082A (en) | Porcine pseudorabies virus detection test paper | |
| CN102520169A (en) | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof | |
| CN104109206A (en) | Monoclonal antibodies against duck Tembusu virus, antigen detection kit and application | |
| CN103235119A (en) | A diagnosis antigen for Toxoplasma gondii infection and a preparation method and applications thereof | |
| Tarigan et al. | Characterization of the M2e antibody response following highly pathogenic H5N1 avian influenza virus infection and reliability of M2e ELISA for identifying infected among vaccinated chickens | |
| CN101216492A (en) | Test paper for rapidly detecting pseudorabies antibody and method for making same | |
| CN103698514A (en) | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting lawsonia intracellularis antibody | |
| Ghanem et al. | Prevalence and risk factors of caprine arthritis encephalitis virus infection (CAEV) in Northern Somalia | |
| CN103616509B (en) | Detect E III-indirect ELISA antibody assay kit and the application of Latex agglutination test | |
| CN103364569B (en) | Bovine Cryptosporidium ELISA detection kit | |
| CN105061602A (en) | Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof | |
| CN215728195U (en) | Novel coronavirus SARS-CoV-2 antibody detection test paper strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150225 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |