CN105301240A - Duck parvovirus indirect ELISA (enzyme-linked immuno sorbent assay) detection reagent kit - Google Patents

Duck parvovirus indirect ELISA (enzyme-linked immuno sorbent assay) detection reagent kit Download PDF

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Publication number
CN105301240A
CN105301240A CN201510603584.6A CN201510603584A CN105301240A CN 105301240 A CN105301240 A CN 105301240A CN 201510603584 A CN201510603584 A CN 201510603584A CN 105301240 A CN105301240 A CN 105301240A
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duck
duck parvovirus
protein
parvovirus
solution
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刁有祥
陈浩
窦砚国
冯强
郑肖强
牛晓宇
杨晶
于相龙
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Shandong Agricultural University
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Shandong Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus

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  • Immunology (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the technical field of veterinary pathogeny antibody test, in particular to a duck parvovirus indirect ELISA (enzyme-linked immuno sorbent assay) detection reagent kit. The inventor firstly provides a duck parvovirus virus VP3 protein; the amino acid sequence of the protein is shown as SEQ ID No.1; the amino acid sequence expressing the protein is shown as SEQ ID No.2. Study shows that the protein has good immune response original performance, and can be used for DPV (duck plague virus) antibody detection; further, the protein is used for providing the duck parvovirus indirect ELISA detection reagent kit. The reagent kit has the advantages of high speed, high stability, high specificity and high sensitivity, can be used for fast detecting the antibody level of the duck parvovirus virus in the serum, and achieves the obvious promotion effect on the prevention and the control of the disease.

Description

A kind of Duck parvovirus indirect ELISA testing kit
Technical field
The present invention relates to animal doctor's pathogenic autoantibody detection technique field, provide a kind of Duck parvovirus indirect ELISA testing kit.
Background technology
Duck parvovirus is the one of parvovirus, mainly encroaches on the duckling in 1 ~ 3 week age, clinical soft for cardinal symptom with diarrhoea, expiratory dyspnea and pin, all there is generation throughout the year, raise to duck and bring great economic loss, be one of main disease during duck is raised, M & M is also higher.
Since in March, 2015, there is a kind of disease in the ground meat duck groups such as China Shandong, Jiangsu and Anhui, empirical tests is Duck parvovirus, this disease is with the depauperation of duck beak, and tongue is overhanging is feature, and this disease mainly occurs in the commodity duck of more than 14 ages in days, the duck group incidence of disease is 10%-20%, reach as high as 50%, jumpbogroup infection rate reaches as high as 100%, but this disease does not cause trouble duck dead.Suffer from duck hypoevolutism, body temperature slightly raises, and beak is short, and elasticity reduces, and swollem tongue is overhanging, and the infection later stage suffers from duck shin bone and wing bone is easily fractured.This disease mainly causes duck group feedstuff-meat ratio to rise, and aquaculture cost raises, and duck is delivered qualification rate for sale and reduces.Huge economic loss is caused to meat duck aquaculture.
Parvovirus belongs to (Parvovirus) to be a kind of nucleic acid is the DNA virus of sub-thread wire, and virus structure polypeptide has 3 kinds, is respectively VP1, VP2, VP3, and wherein VP3 is main structural polypeptide.This virus can not aggegation chicken red blood cell; it is the major protein forming parvovirus outside; the neutrality epitope resisted DPV containing protecting body and infect, utilizes and sets up ELISA detection kit containing the recombinant viral proteins of Neutralization and crystallization and have great importance for the diagnosis of this disease.
Summary of the invention
For above-mentioned situation, the present inventor provides a kind of Duck parvovirus indirect ELISA testing kit, and inventor provide firstly a kind of VP3 albumen of Duck parvovirus, and this protein amino acid sequence is as shown in SEQIDNo.1, express the nucleotide sequence of this albumen, as shown in SEQIDNo.2.Research shows, this albumen has good immune response originality, may be used for the detection of DPV antibody, and then utilize this albumen to provide Duck parvovirus antibody ELISA detection kit, this kit has fast, stablize, specificity is high, sensitivity is strong, can detect the antibody horizontal of Duck parvovirus in serum fast, the prevention and corntrol for this disease has obvious impetus.
Inventor provide firstly a kind of VP3 albumen of Duck parvovirus, and this protein amino acid sequence, as shown in SEQIDNo.1, expresses the nucleotide sequence of this albumen, as shown in SEQIDNo.2.Research shows, this albumen has good immune response originality, may be used for the detection of DPV antibody.
On this basis, inventor obtains Duck parvovirus indirect ELISA testing kit, and described kit comprises: the elisa plate of Duck parvovirus VP3 recombinant protein bag quilt, ELIAS secondary antibody, positive control, negative control, cleansing solution, sample diluting liquid, nitrite ion and stop buffer;
Wherein ELIAS secondary antibody is horseradish peroxidase mark goat-anti duck monoclonal antibody, and described positive control is natural infection Duck parvovirus GT2015 strain duck serum; Described negative control is normal duck serum, and described restructuring VP3 albumen package amount is 150ng/well.
Described coating buffer, cleansing solution, confining liquid, sample diluting liquid, component and the proportioning of nitrite ion and stop buffer are as follows:
Coating buffer: 1 × carbonate buffer solution: Na 2cO 30.2756g, NaHCO 30.6216g, dissolves with distilled water and is settled to 100mL, pH value 9.5-9.7,4 DEG C of preservations;
PBS solution: NaCl4.25g, NaH 2pO 42H 2o0.178g, Na 2hPO 412H 2o1.386g, dissolves with distilled water and is settled to 500mL, pH value 7.1-7.3;
Cleansing solution: every 1LPBS adds Tween-200.5mL, fully mixes;
Confining liquid, dilution: be dissolved in by 5gdrymilk in 100mLPBST dilution, short-term preservation, in 4 DEG C, is stored in-20 DEG C for a long time;
ELIAS secondary antibody: Peroxidase-LabeledAntibodyToDuckIgG (H+L), ProducedinGoat;
TMB nitrite ion: soluble type single component tmb substrate solution;
Stop buffer is 3mol/L sulfuric acid solution.
The recombinant protein preparation of the wherein said Duck parvovirus VP3 albumen as ELISA Plate envelope antigen, comprise the following steps: utilize the viral DNA in the sick duck tissue of phenol chloroform method extracting, detect with round pcr, pcr amplified fragment (SEQIDNo.2) is carried out glue and reclaim purifying, PET-28a (+) carrier is proceeded to after restriction enzyme BamHI and HindIII double digestion, be converted in DH5 α competent escherichia coli cell, be accredited as positive colony through bacterium liquid PCR, plasmid order-checking is extracted in concussion cultivation for 16 hours.Positive plasmid is proceeded in BL21 competent escherichia coli cell, what be accredited as the positive carries out IPTG abduction delivering, carry out washing with urea method to purify, then verify albumen (SEQIDNo.1) purification result and immunogenicity result with SDS-PAGE and Westernblot respectively.Result display object band is the Duck parvovirus VP3 albumen of abduction delivering of the present invention.
Inventor further provides the using method of above-mentioned Duck parvovirus indirect ELISA testing kit, specific as follows:
1, ELISA trace routine
(1) recombinate Duck parvovirus VP3 albumen bag by ELISA reaction plate with 150ng/well, 4 DEG C of night incubation, application PBST washs ELISA reaction plate 3 times;
(2) close ELISA reaction plate with the skim milk powder solution of 5g/100mL, 37 DEG C, after closing 1h, application PBST washs ELISA reaction plate 3 times;
(3) serum 5g/100mL skim milk powder solution to be checked is done 1:10 times of w/v dilution, add ELISA reaction plate by 100 μ L, after 37 DEG C of effect 1.5h, application PBST washs ELISA reaction plate 3 times;
(4) goat-anti duck IgG-HRP is added ELISA reaction plate by 100 μ L/well, after 37 DEG C of effect 1h, application PBST washs ELISA reaction plate 3 times;
(5) 100 μ L substrate nitrite ions are added ELISA reaction plate, lucifuge 37 DEG C colour developing 15min, adds 50 μ L stop buffers, reads OD value immediately under microplate reader 450nm wavelength.
2, the determination of indirect ELISA method criterion
The OD450nm value of ELISA Plate is read by microplate reader, calculate negative serum mean value (`X)+standard deviation (SD), obtaining yin and yang attribute critical value is 0.239, result criterion is X >=0.25, the positive can be judged as, namely contain Duck parvovirus antibody in measuring samples, otherwise be negative.
In sum, Duck parvovirus indirect ELISA testing kit provided by the invention and using method thereof have fast, stablize, specificity is high, sensitivity is strong, can detect the antibody horizontal of Duck parvovirus in serum fast, the prevention and corntrol for this disease has obvious impetus.
Preservation information
The preservation time: on August 28th, 2015
Depositary institution's title: China typical culture collection center CCTCC
Deposit number: CCTCCNO:V201527
Depositary institution address: Wuhan, China university
Classification And Nomenclature: Duck parvovirus GT2015
Accompanying drawing explanation
Fig. 1 is the recombinant protein SDS-PAGE electrophoretogram of Duck parvovirus VP3 albumen,
In figure, M is Blue iIProteinMarker, 1 is non-abduction delivering recombinant protein, and 2 is 4h abduction delivering recombinant protein, and 3 is recombinant protein after purifying,
Non-inducible protein 1 can be shown there is no destination protein from figure, only have part e. coli protein; Induce non-purifying protein 2 to have destination protein, but also have the albumen of a lot of Escherichia coli self simultaneously; And the recombinant protein 3 of purifying only has destination protein not have other foreign proteins.
Embodiment
The acquisition of embodiment 1 purification of Recombinant Duck parvovirus VP3 albumen
The extraction of A, viral DNA: after getting the digestion of part trouble duck liver homogenate suspension, adopt phenol chloroform method to carry out the extraction of STb gene, finally use 50 μ LddH 2o dissolves, and is placed in-20 DEG C of preservations;
The Design and synthesis of B, primer: according to the Duck parvovirus whole genome sequence delivered, in conjunction with us to many strains Duck parvovirus separated strain VP3 sequencing result, utilize PrimerPremier5.0 to design 1 pair of primer and add restriction enzyme BamHI and HindIII restriction enzyme site, its nucleotide sequence of F1:5 '-CGGATCCAAGGAAGTCACAACGCAGGAT-3 ' is as shown in SEQIDNo.3, and its nucleotide sequence of R1:5 '-CAAGCTTGCCATCAGTCTTCGGTATT-3 ' is as shown in SEQIDNo.4;
C, pcr amplification: PCR reaction system is: 2 × EsTaqMasterMix10 μ L, upstream and downstream primers F 1/R1 (20 μm of ol/L) each 1 μ L, 1 μ L viral DNA template, adds ddH 2o is 20 μ L to cumulative volume.
PCR reaction conditions: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 30 circulations; Last 72 DEG C extend 10min;
The preparation of D, positive plasmid standard items: by pcr amplification product after the agarose gel electrophoresis of 1%, cuts object band at 921bp place, reclaims with glue the purifying that kit carries out target DNA; Purified product is connected to pMD18-T carrier, transforms DH5 α competent escherichia coli cell, picking positive colony, and 18h is cultivated in 37 DEG C of concussions, serves the order-checking of Hai Shenggong bioengineering company limited.Adopt plasmid Mini Kit to extract the correct clone plasmid of order-checking, finally use 50 μ L cleansing solution wash-outs;
The abduction delivering of E, recombinant plasmid and purifying: the plasmid of extraction is proceeded to BL21 competent escherichia coli cell and be accredited as and positive carry out IPTG abduction delivering, carry out washing with urea method and purify;
F, SDS-PAGE protein electrophorese: the recombinant protein of purifying is carried out SDS-PAGE protein electrophoresis, whether checking is the recombinant protein of expressing, the recombinant protein of result Explicit Expression is the VP3 albumen of Duck parvovirus, and this protein amino acid sequence is as shown in SEQIDNo.1.
The assembling of embodiment 2 Duck parvovirus indirect ELISA testing kit
Recombinant protein bag quilt: the recombinant protein of purifying is quantitatively surveyed its concentration, gets appropriate recombinant protein and mixes in 10mL1 × carbonate buffer solution, makes its final concentration be 1.5ng/L, then adds at the bottom of 96 ELISA Plate holes, hole, every hole 100 μ L.Cover shrouding film, 4 DEG C of overnight incubation.
Kit comprises following reagent: 96 hole ELISA Plate 1 piece, 30 × PBST cleansing solution 20mL, 1 × carbonate buffer solution (PH=9.6) 10mL, skimmed milk power 2.5g, GoatAnti-DuckIgG-HRP10mL, soluble type single component tmb substrate nitrite ion 10mL, stop buffer (3mol/LH 2sO 4) 10mL, shrouding film 4.Preserve 6 months for 2-8 DEG C
Wherein said coating buffer, cleansing solution, confining liquid, sample diluting liquid, component and the proportioning of nitrite ion and stop buffer are as follows:
Coating buffer: 1 × carbonate buffer solution: Na 2cO 30.2756g, NaHCO 30.6216g, dissolves with distilled water and is settled to 100mL, pH value 9.5-9.7,4 DEG C of preservations;
PBS solution: NaCl4.25g, NaH 2pO 42H 2o0.178g, Na 2hPO 412H 2o1.386g, dissolves with distilled water and is settled to 500mL, pH value 7.1-7.3;
Cleansing solution: every 1LPBS adds Tween-200.5mL, fully mixes;
Confining liquid, dilution: be dissolved in by 5gdrymilk in 100mLPBST dilution, short-term preservation, in 4 DEG C, is stored in-20 DEG C for a long time;
ELIAS secondary antibody: Peroxidase-LabeledAntibodyToDuckIgG (H+L), ProducedinGoat;
TMB nitrite ion: soluble type single component tmb substrate solution;
Stop buffer is 3mol/L sulfuric acid solution.
The using method of embodiment 3 Duck parvovirus indirect ELISA testing kit
Described using method concrete steps are as follows:
1, ELISA trace routine
(1) recombinate Duck parvovirus VP3 albumen bag by ELISA reaction plate with 150ng/well, 4 DEG C of night incubation, application PBST washs ELISA reaction plate 3 times;
(2) close ELISA reaction plate with the skim milk powder solution of 5g/100mL, 37 DEG C, after closing 1h, application PBST washs ELISA reaction plate 3 times;
(3) serum 5g/100mL skim milk powder solution to be checked is done 1:10 times of w/v dilution, add ELISA reaction plate by 100 μ L, after 37 DEG C of effect 1.5h, application PBST washs ELISA reaction plate 3 times;
(4) goat-anti duck IgG-HRP is added ELISA reaction plate by 100 μ L/well, after 37 DEG C of effect 1h, application PBST washs ELISA reaction plate 3 times;
(5) 100 μ L substrate nitrite ions are added ELISA reaction plate, lucifuge 37 DEG C colour developing 15min, adds 50 μ L stop buffers, reads OD value immediately under microplate reader 450nm wavelength.
2, the determination of indirect ELISA method criterion
The OD450nm value of ELISA Plate is read by microplate reader, calculate negative serum mean value (`X)+standard deviation (SD), obtaining yin and yang attribute critical value is 0.239, result criterion is X >=0.25, the positive can be judged as, namely contain Duck parvovirus antibody in measuring samples, otherwise be negative.

Claims (3)

1. a Duck parvovirus indirect ELISA testing kit, it is characterized in that: the envelope antigen adopted is the restructuring VP3 albumen of Duck parvovirus, this protein amino acid sequence, as shown in SEQIDNo.1, expresses the nucleotide sequence of this albumen as shown in SEQIDNo.2.
2. Duck parvovirus indirect ELISA testing kit according to claim 1, it is characterized in that: described kit comprises: the elisa plate of Duck parvovirus VP3 recombinant protein bag quilt, ELIAS secondary antibody, positive control, negative control, cleansing solution, sample diluting liquid, substrate solution A, substrate solution B and stop buffer;
Wherein ELIAS secondary antibody is horseradish peroxidase mark goat-anti duck monoclonal antibody; Described positive control is natural infection Duck parvovirus GT2015 strain duck serum; Described negative control is normal duck serum, and described restructuring VP3 albumen package amount is 150ng/well.
3. Duck parvovirus indirect ELISA testing kit according to claim 2, is characterized in that: described coating buffer, cleansing solution, confining liquid, sample diluting liquid, and component and the proportioning of nitrite ion and stop buffer are as follows:
Coating buffer: 1 × carbonate buffer solution: Na 2cO 30.2756g, NaHCO 30.6216g, dissolves with distilled water and is settled to 100mL, pH value 9.5-9.7,4 DEG C of preservations;
PBS solution: NaCl4.25g, NaH 2pO 42H 2o0.178g, Na 2hPO 412H 2o1.386g, dissolves with distilled water and is settled to 500mL, pH value 7.1-7.3;
Cleansing solution: every 1LPBS adds Tween-200.5mL, fully mixes;
Confining liquid, dilution: be dissolved in by 5gdrymilk in 100mLPBST dilution, short-term preservation, in 4 DEG C, is stored in-20 DEG C for a long time;
ELIAS secondary antibody: Peroxidase-LabeledAntibodyToDuckIgG (H+L), ProducedinGoat;
TMB nitrite ion: soluble type single component tmb substrate solution;
Stop buffer is 3mol/L sulfuric acid solution.
CN201510603584.6A 2015-09-21 2015-09-21 Duck parvovirus indirect ELISA (enzyme-linked immuno sorbent assay) detection reagent kit Pending CN105301240A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956362A (en) * 2021-09-01 2022-01-21 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation

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FR2741631A1 (en) * 1995-11-23 1997-05-30 Cneva Laboratoire Central De R DNA encoding duck parvovirus capsid proteins
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FR2741631A1 (en) * 1995-11-23 1997-05-30 Cneva Laboratoire Central De R DNA encoding duck parvovirus capsid proteins
CN101914499A (en) * 2010-08-03 2010-12-15 中国农业科学院哈尔滨兽医研究所 Anti-waterfowl parvovirus VP3 protein monoclonal antibody
WO2012031760A1 (en) * 2010-09-08 2012-03-15 Medigene Ag Parvovirus mutated structural proteins comprising cross - protective b - cell epitopes of a hpv l2 protein as well as products and methods relating thereto

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956362A (en) * 2021-09-01 2022-01-21 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation
CN113956362B (en) * 2021-09-01 2022-09-06 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation

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